Internalizing ErbB2 antibodies

ABSTRACT

This invention provides novel erbB2-binding internalizing antibodies. The antibodies, designated F5 and C1, specifically bind to c-erbB2 antigen and, upon binding, are readily internalized into the cell bearing the c-erbB2 marker. Chimeric molecules comprising the F5 and/or C1 antibodies attached to one or more effector molecules are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of provisionalapplication U.S. Ser. No. 60/082,953, filed on Apr. 24, 1998, which isherein incorporated by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported, in part, by Department of Defense GrantsDAMD17-96-1-6244 and DAMD17-94-4433. The government of the United Statesof America may have some rights in this invention.

FIELD OF THE INVENTION

This invention pertains to the fields of immunodiagnostics andimmunotherapeutics. More particularly this invention pertains to thediscovery of novel human antibodies that specifically bind c-erbB-2, andto chimeric molecules containing these antibodies.

BACKGROUND OF THE INVENTION

It is generally desired to identify antibodies that specifically bind toparticular classes of cells (e.g. tumor cells). Such antibodies,sometimes referred to as “targeting antibodies”, can be used tospecifically direct (deliver) various effector molecules (e.g.liposomes, cytotoxins, labels, etc.) to the target cell.

Targeting antibodies have been of particular interest in the study andtreatment of cancer. A major goal of cancer research has been toidentify tumor antigens that are qualitatively or quantitativelydifferent from normal cells (Goldenberg (1994) Ca: A Cancer J. forClinicians. 44: 43-64). The presence and/or quantity of such antigenscould be detected by antibodies and such detection forms the basis ofdiagnostic and prognostic tests. In addition, the antibodies could beused to selectively kill tumor cells either directly via their effectorfunction (Brown et al. (1989) Blood. 73: 651-661) or by attachingcytotoxic molecules to the antibody (Vitetta et al. (1987) Science. 238:1098-1104; Brinkmann et al. (1993) Proc. Natl. Acad. Sci. USA. 90:547-551).

Despite the demonstration of antigens that are overexpressed on tumorcells, antibodies have been used with limited success for diagnosis andtreatment of solid tumors, (see review in Riethmuller et al. (1992)Curr. Opin. Immunol. 4: 647-655, and Riethmuller (1993) Curr. OpinionImmunol. 5: 732-739). Their utility has been hampered by the paucity oftumor specific antibodies, antibody immunogenicity, low bindingaffinity, and poor tumor penetration.

Nonspecific toxicity results from the failure of the antibody to bindspecifically and with high affinity to tumor cells. As a result,nonspecific cell killing occurs. In addition, the foreign immunotoxinmolecule elicits a strong immune response in humans. The immunogenicityof the toxin portion of the immunotoxin has recently been overcome byusing the human analogue of RNase (Rybak et al. (1992) Proc. Nat. Acad.Sci., USA, 89: 3165). The murine antibody portion, however, is stillsignificantly immunogenic (Sawler et al., (1995) J. Immunol., 135:1530).

Immunogenicity could be avoided and toxicity reduced if high affinitytumor specific human antibodies were available. However, the productionof human monoclonal antibodies using conventional hybridoma technologyhas proven extremely difficult (James et al., (1987) J. Immunol. Meth.,100: 5). Furthermore, the paucity of purified tumor-specific antigensmakes it necessary to immunize with intact tumor cells or partiallypurified antigen. Most of the antibodies produced react with antigensthat are also common to normal cells and are therefore unsuitable foruse as tumor-specific targeting molecules.

SUMMARY OF THE INVENTION

This invention provides two new internalizing anti-c-erbB-2 antibodiesdesignated herein as F5 and C1 respectively. Preferred antibodiesspecifically bind to a c-erbB2 receptor and are antibody F5-derived orC1-derived antibodies (i.e., antibodies that bind to a c-erbB2 receptorepitope bound by F5 and/or C1). The antibodies preferably comprise anamino acid sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 1 having conservative substitutions, and SEQ IDNO: 2 having conservative substitutions. Particularly preferredantibodies share at least 70% sequence identity with the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2 and have a binding affinity for−erbB2 on cells of at least 10⁵ M. In one embodiment the antibodies willhave an amino acid sequence that differs from the amino acid sequence ofSEQ ID NO: 1 or SEQ ID NO: 2 by no more than 30 residues. The antibodycan comprise at least one, at least two or at least three of thecomplementarity determining regions (CDRs) of SEQ ID NO: 1 and/or SEQ IDNO: 2. In addition, or alternatively, the antibody can comprise at leastone, at least two, or at least three framework regions of SEQ ID NO: 1and/or SEQ ID NO: 2. Particularly preferred F5 and C1 antibodies havethe amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

In another embodiment, this invention provides for antibodies that arecross-reactive with an anti-idiotypic antibody raised against F5 or C1.Thus, this invention provides for an antibody that specifically binds toa c-erbB2 receptor, where the antibody comprises at least 10 contiguousamino acids from the polypeptide sequence as set forth in SEQ ID NO: 1or SEQ ID NO: 2, and where the antibody, when presented as an antigen,elicits the production of an anti-idiotypic antibody that specificallybinds to a polypeptide sequence as set forth in SEQ ID NO: 1 or SEQ IDNO: 2; and the antibody does not bind to antisera raised against thepolypeptide set forth in SEQ ID NO: 1 and SEQ ID NO: 2, that has beenfully immunosorbed with the polypeptides set forth in SEQ ID NO: 1 andin SEQ ID NO: 2. These antibodies can share at least 70% sequenceidentity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2and have a binding affinity for −erbB2 on cells of at least 10:M. Theantibody may comprise an amino acid sequence that differs from the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 by no more than 30residues. The antibody may comprises a complementarity determiningregion (CDR) of SEQ ID NO: 1 or SEQ ID NO: 2.

This invention also provides for the epitopes specifically recognized byF5 or C1. These are easily identified by epitope mapping methodsutilizing the F5 or C1 antibodies provided herein.

In still another embodiment, this invention provides methods ofspecifically delivering an effector molecule to a cell bearing a c-erbB2receptor. The methods involve providing a chimeric molecule comprisingthe effector molecule attached to any of the C1-derived or F5-derivedantibodies described herein and contacting the cell bearing a c-erbB2with the chimeric molecule, whereby the chimeric molecule specificallybinds to the cell. In a particularly preferred embodiment, all, or aportion of the chimeric molecule is internalized into the cell.Preferred effector molecules include, but are not limited to,cytotoxins, labels, radionuclides, drugs, liposomes, ligands,antibodies, and the like. In a particularly preferred embodiment, theeffector molecule is a protein or contains a protein and the chimericmolecule is a fusion protein. Particularly preferred target cells arecancer (e.g. metastatic or solid tumor, e.g. breast cancer) cells

This invention also provides for the chimeric molecules that compriseany of the antibodies described herein attached to any of the effectormolecules described herein. Preferred chimeric molecules bind a cellbearing a c-erbB-2. Particularly preferred chimeric molecules are fusionproteins.

This invention also provides for nucleic acids encoding the variousconstructs described herein. Thus, in one embodiment, this inventionprovides nucleic acids encode an antibody that specifically binds to ac-erbB2 receptor, where the antibody is an F5-derived or a C1-derivedantibody that specifically binds to the epitope bound by F5 (SEQ IDNO: 1) or C1 (SEQ ID NO: 2). Preferred nucleic acids encode an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 1 having conservative substitutions, and SEQ ID NO: 2having conservative substitutions. Particularly preferred nucleic acidshybridize with a nucleic acid that encodes the antibodies of SEQ ID NO:1 or SEQ ID NO: 2 under stringent conditions. Other preferred nucleicacids encode an antibody that shares at least 70% sequence identity withthe amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and wherein saidantibody has a binding affinity for −erbB2 on cells of at least 10 μM.Particularly preferred nucleic acids encodes an amino acid sequence thatdiffers from the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 byno more than 30 residues. Another preferred nucleic acid encodes one ormore complementarity determining regions (CDRs) of SEQ ID NO: 1 or SEQID NO: 2. Other preferred nucleic acids comprise a sequence encoding theamino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Where the chimeric proteins are fusion proteins this invention alsoprovides nucleic acids encoding the chimeric proteins.

In another embodiment, this invention provides for cells (e.g.eukaryotic or prokaryotic) expressing any of the nucleic acids describedherein.

This invention also provides for pharmaceutical compositions. Thepharmaceutical compositions preferably comprise a pharmacologicalexcipient and one or more of the F5-derived or C-derived antibodiesdescribed herein.

Kits are also provides as described herein for the diagnosis andtreatment of pathological conditions (e.g. cancers) or for the practiceof any of the screening or transfection methods described herein.

DEFINITIONS

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies (antibodies that exist as a single polypeptide chain), morepreferably single chain Fv antibodies (scFv or scFv) in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide. The singlechain Fv antibody is a covalently linked V_(H)-V_(L) heterodimer whichmay be expressed from a nucleic acid including V_(H)- and V_(L)-encodingsequences either joined directly or joined by a peptide-encoding linker.Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Whilethe V_(H) and V_(L) are connected to each as a single polypeptide chain,the V_(H) and V_(L) domains associate non-covalently. The firstfunctional antibody molecules to be expressed on the surface offilamentous phage were single-chain Fv's (scFv), however, alternativeexpression strategies have also been successful. For example Fabmolecules can be displayed on phage if one of the chains (heavy orlight) is fused to g3 capsid protein and the complementary chainexported to the periplasm as a soluble molecule. The two chains can beencoded on the same or on different replicons; the important point isthat the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to g3p (see, e.g., U.S. Pat.No. 5,733,743). The scFv antibodies and a number of other structuresconverting the naturally aggregated, but chemically separated light andheavy polypeptide chains from an antibody V region into a molecule thatfolds into a three dimensional structure substantially similar to thestructure of an antigen-binding site are known to those of skill in theart (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778).Particularly preferred antibodies include all those that have beendisplayed on phage I think preferred antibodies should include all thathave been displayed on phage (e.g., scFv, Fv, Fab and disulfide linkedFv (Reiter et al. (1995) Protein Eng. 8: 1323-1331).

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

An “internalizing antibody” is an antibody that, upon binding to areceptor or other ligand on a cell surface is transported into the cell(e.g. into a vacuole or other organelle or into the cytoplasm of thecell.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smaller Kdrepresents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and on geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (k_(on)) andthe “off rate constant” (k_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.The ratio of k_(off)/k_(on) enables cancellation of all parameters notrelated to affinity and is thus equal to the dissociation constant K_(d)(see, generally, Davies et al. (1990) Ann. Rev. Biochem., 59: 439-473.

The phrase “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein and do not bind in a significant amount toother proteins present in the sample. Specific binding to a proteinunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, F5 or C1 antibodiescan be raised to the c-erbB-2 protein that bind c-erbB-2 and not toother proteins present in a tissue sample. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. The amino acidresidues are preferably in the natural “L” isomeric form. However,residues in the “D” isomeric form can be substituted for any L-aminoacid residue, as long as the desired functional property is retained bythe polypeptide. In addition, the amino acids, in addition to the 20“standard” amino acids, include modified and unusual amino acids, whichinclude, but are not limited to those listed in 37 CFR §1.822(b)(4).Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates either a peptide bond to afurther sequence of one or more amino acid residues or a covalent bondto a carboxyl or hydroxyl end group.

The term “binding polypeptide” refers to a polypeptide that specificallybinds to a target molecule (e.g. a cell receptor) in a manner analogousto the binding of an antibody to an antigen. Binding polypeptides aredistinguished from antibodies in that binding polypeptides are notultimately derived from immunoglobulin genes or fragments ofimmunoglobulin genes.

The terms “F5 antibody” or “C1 antibody” typically refer to antibodiesthat bind to the epitope(s) bound by F5 or C1 respectively. Preferred F5or C1 antibodies are internalizing antibodies. F5 and C1 when used torefer to the prototypical antibody refer to antibodies having thesequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The F5 antibodyis also referred to as 3TF5 in the examples.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated.

Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka etal. (1985) J. Biol. Chem. 260: 2605-2608; and Cassol et al. (1992);Rossolini et al., (1994) Mol. Cell. Probes 8: 91-98). The term nucleicacid is used interchangeably with gene, cDNA, and mRNA encoded by agene.

The terms “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state. However, the term “isolated”is not intended refer to the components present in an electrophoreticgel or other separation medium. An isolated component is free from suchseparation media and in a form ready for use in another application oralready in use in the new application/milieu.

The terms “identical” or percent “identity,” or percent “homology” inthe context of two or more nucleic acids or polypeptide sequences, referto two or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence, as measuredusing one of the following sequence comparison algorithms or by visualinspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90-95% or evenat least 98% amino acid residue identity across a window of at least 30nucleotides, preferably across a window of at least 40 nucleotides, morepreferably across a window of at least 80 nucleotides, and mostpreferably across a window of at least 100 nucleotides, 150 nucleotides,200 nucleotides or greater, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins & Sharp (1989) CABIOS 5:151-153. The program can align up to 300sequences, each of a maximum length of 5,000 nucleotides or amino acids.The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al. (1990) J. Mol. Biol. 215:403-410.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extension of the word hits in each direction are halted when:the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or the end of either sequence is reached. The BLAST algorithm parametersW, T, and X determine the sensitivity and speed of the alignment. TheBLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci.USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4,and a comparison of both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

The phrases “hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refer to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2 “Overview of principles of hybridization and thestrategy of nucleic acid probe assays”, Elsevier, New York. Generally,highly stringent hybridization and wash conditions are selected to beabout 5° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T_(m) for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.) suprafor a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Anexample medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratio of2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

The terms F5 and C1 when referring to antibodies refer to the antibodiesof SEQ ID NOS: 1 and 2, respectively. Preferred F5 and C1 antibodiesadditionally include the antibodies of SEQ ID NOS: 1 and 2,respectively, or conservative substitutions of these sequences so longas the binding specificity of F5 and C1 is preserved. The F5-derived andC1-derived antibodies are antibodies derived sequences (from the aminoacid or nucleic acid) of F5 or C1, respectively. The F5- and C1-derivedantibodies are preferably obtained by one of several types of derivationthat include, but are not limited to: 1) derivation by chain shuffling;2) derivation by site directed mutagenesis of CDRs; 3) derivation byintroducing multiple mutations into sequence by either error prone PCR,mutator strains of E. coli, or “DNA shuffling (Crameri et al. (1996)Nature Medicine. 2: 100-102).” The F5 and C1 derived antibodies arerecognized by their cross-reactivity with either 1) the c-erbB-2 epitoperecognized by F5 and C1; and/or 2) An anti F5 or C1 anti-idiotypicantibody. F5 and C1 antibodies preferably have a binding affinity ofabout 2:M, more preferably of less than about 1:M, and most preferablyof less than about 100 nM or better and are preferably derived byscreening (for affinity the epitopes bound by F5 and C1, respectively) aphage display library in which a known F5 or C1 variable heavy (V_(H))chain is expressed in combination with a multiplicity of variable light(V_(L)) chains or conversely a known F5 or C1 variable light chain isexpressed in combination with a multiplicity of variable heavy (V_(H))chains. F5- or C1-derived antibodies also include those antibodiesproduced by the introduction of mutations into the variable heavy orvariable light complementarity determining regions (CDR1, CDR2 or CDR3)as described herein. Finally F5 and C1 antibodies include thoseantibodies produced by any combination of these modification methods asapplied to F5 or C1 and their derivatives.

A chimeric molecule is a molecule in which two or more molecules thatexist separately in their native state are joined together to form asingle molecule having the desired functionality of all of itsconstituent molecules. While the chimeric molecule may be prepared bycovalently linking two molecules each synthesized separately, one ofskill in the art will appreciate that where the chimeric molecule is afusion protein, the chimera may be prepared de novo as a single “joined”molecule.

A fusion protein is a chimeric molecule in which the constituentmolecules are all polypeptides and are attached (fused) to each otherthrough terminal peptide bonds so that the chimeric molecule is acontinuous single-chain polypeptide. The various constituents can bedirectly attached to each other or can be coupled through one or morepeptide linkers.

An effector moiety or molecule is a molecule or moiety that typicallyhas a characteristic activity that is desired to be delivered to atarget cell (e.g. a tumor overexpressing c-erbB-2). Effector moleculesinclude cytotoxins, labels, radionuclides, ligands, antibodies, drugs,liposomes, and viral coat proteins that render the virus capable ofinfecting a c-erbB-2 expressing cell.

A “target” cell refers to a cell or cell-type that is to be specificallybound by a member of a phage display library or a chimeric molecule ofthis invention. Preferred target cells are cells for which aninternalizing antibody or binding polypeptide is sought. The target cellis typically characterized by the expression or overexpression of atarget molecule that is characteristic of the cell type. Thus, forexample, a target cell can be a cell, such as a tumor cell, thatoverexpresses a marker such as c-erbB-2.

A “targeting moiety” refers to a moiety (e.g. a molecule) thatspecifically binds to the target molecule. Where the target molecule isa molecule on the surface of a cell and the targeting moiety is acomponent of a chimeric molecule, the targeting moiety specificallybinds the chimeric molecule to the cell bearing the target. Where thetargeting moiety is a polypeptide it can be referred to as a “targetingpolypeptide”.

The terms “internalizing” or “internalized” when used in reference to acell refer to the transport of a moiety (e.g. phage) from outside toinside a cell. The internalized moiety can be located in anintracellular compartment, e.g. a vacuole, a lysosome, the endoplasmicreticulum, the golgi apparatus, or in the cytosol of the cell itself.

An internalizing receptor or marker is a molecule present on theexternal cell surface that when specifically bound by an antibody orbinding protein results in the internalization of that antibody orbinding protein into the cell. Internalizing receptors or markersinclude receptors (e.g., hormone, cytokine or growth factor receptors)ligands and other cell surface markers binding to which results ininternalization.]

The term “heterologous nucleic acid” refers to a nucleic acid that isnot native to the cell in which it is found or whose ultimate origin isnot the cell or cell line in which the “heterologous nucleic acid” iscurrently found.

The idiotype represents the highly variable antigen-binding site of anantibody and is itself immunogenic. During the generation of anantibody-mediated immune response, an individual will develop antibodiesto the antigen as well as anti-idiotype antibodies, whose immunogenicbinding site (idiotype) mimics the antigen. Anti-idiotypic antibodiescan also be generated by immunization with an antibody, or fragmentthereof.,

A “phage display library” refers to a collection of phage (e.g.,filamentous phage) wherein the phage express an external (typicallyheterologous) protein. The external protein is free to interact with(bind to) other moieties with which the phage are contacted. Each phagedisplaying an external protein is a “member” of the phage displaylibrary.

The term “filamentous phage” refers to a viral particle capable ofdisplaying a heterogenous polypeptide on its surface. Although oneskilled in the art will appreciate that a variety of bacteriophage maybe employed in the present invention, in preferred embodiments thevector is, or is derived from, a filamentous bacteriophage, such as, forexample, fl, fd, Pfl, M13, etc. The filamentous phage may contains aselectable marker such as tetracycline (e.g., “fd-tet”). Variousfilamentous phage display systems are well known to those of skill inthe art (see, e.g., Zacher et al. (1980) Gene 9: 127-140, Smith et al.(1985) Science 228: 1315-1317 (1985); and Parmley and Smith (1988) Gene73: 305-318).

A “viral packaging signal” is a nucleic acid sequence necessary andsufficient to direct incorporation of a nucleic acid into a viralcapsid.

An assembly cell is a cell in which a nucleic acid can be packaged intoa viral coat protein (capsid). Assembly cells may be infected with oneor more different virus particles (e.g. a normal or debilitated phageand a helper phage) that individually or in combination direct packagingof a nucleic acid into a viral capsid.

The following abbreviations are used herein: AMP, ampicillin; c-erbB-2ECD, extracellular domain of c-erbB-2; CDR, complementarity determiningregion; ELISA, enzyme linked immunosorbent assay; FACS, fluorescenceactivated cell sorter; FR, framework region; Glu, glucose; HBS, hepesbuffered saline, 10 mM hepes, 150 mM NaCl, pH 7.4; IMAC, immobilizedmetal affinity chromatography; k_(on), association rate constant;k_(off), dissociation rate constant; MPBS, skimmed milk powder in PBS;MTPBS, skimmed milk powder in TPBS; PBS, phosphate buffered saline, 25mM NaH₂PO₄, 125 mM NaCl, pH 7.0; PCR, polymerase chain reaction; RU,resonance units; scFv or scFv, single-chain Fv fragment; TPBS, 0.05% v/vTween 20 in PBS; SPR, surface plasmon resonance; V_(k), immunoglobulinkappa light chain variable region; V_(λ), immunoglobulin lambda lightchain variable region; V_(L), immunoglobulin light chain variableregion; V_(H), immunoglobulin heavy chain variable region; wt, wildtype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the method for construction of a large human scFvphage antibody library. The strategy for library construction involvedoptimizing the individual steps of library construction to increase boththe efficiency of scFv gene assembly and to increase the efficiency ofcloning assembled scFv genes. (A). First, mRNA from lymphocytes was usedto generate V_(H) and V_(L) gene repertoires by RTPCR which were clonedinto different vectors to create V_(H and V) _(L) gene libraries of8.0×10⁸ and 7.2×10⁶ members respectively. The cloned V-gene librariesprovided a stable and limitless source of V_(H) and V_(L) genes for scFvassembly. DNA encoding the peptide (G₄S)₃ was incorporated into the 5′end of the V_(L) library. This permitted generation of scFv genes by PCRsplicing 2 DNA fragments. Previously, scFv gene repertoires wereassembled from 3 separate DNA fragments consisting of V_(H), V_(L), andlinker DNA. (B) V_(H) and V_(L) gene repertoires were amplified from theseparate libraries and assembled into an scFv gene repertoire usingoverlap extension PCR. The primers used to reamplify the V_(H) and V_(L)gene repertoires annealed 200 bp upstream of the 5′ end of the V_(H)genes and 200 bp down stream of the V_(L) genes. These long overhangsensured efficient restriction enzyme digestion. (C.) The scFv generepertoire was digested with NcoI and NotI and cloned into the plasmidpHEN1 as fusions with the M13 gene III coat protein gene ( ) forphage-display.

FIG. 2 illustrates epitope mapping of F5, C1 and C6.5 scFv. Inhibitionof binding of F5 phage (left panel), C1 phage (center panel) or C6.5phage (right panel) by increasing concentrations of soluble scFv-F5 (o),scFv-C1 (A) or scFv-C6.5 (□). Bound phages were detected with ananti-M13 biotinylated Ab and streptavidin-PE. Results are expressed in %of maximal mean fluorescent intensity (MFI). Soluble F5 and C1 inhibitedthe binding of F5 or C1 phage, but not C6.5 phage.

DETAILED DESCRIPTION I. Introduction.

This invention provides for novel human antibodies that specificallybind to the extracellular domain of the c-erbB-2 protein product of theHER2/neu oncogene. The c-erbB-2 marker is overexpressed by 30-50% ofbreast carcinomas and other adenocarcinomas and thus provides a usefulcell surface marker for specifically targeting tumor cells such ascarcinomas. In contrast to previous known anti-cerbB-2 antibodies, theantibodies of the present invention (designated herein as F5 or C1antibodies) are fully human antibodies. Thus, administration of theseantibodies to a human host elicits a little or no immunogenic response.

In addition, the F5 and C1 antibodies of this invention are rapidlyinternalized into the cell. They are thus extremely useful fordelivering effector moieties into the target cell. Moreover, once aninternalizing antibody or polypeptide is identified it can be used tore-probe one or more cells or cell lines to identify previously unknowninternalizing cellular targets (e.g., receptors).

It is known that other c-erbB2-binding antibodies are not internalized.Thus, without being bound to particular theory, it is believed that theeffective internalization of the F5 and C1 antibodies of this inventionis a consequence of the specific epitope bound by these antibodies. Itis believed that both the F5 and C1 antibody bind the same epitope and,using the F5 and C1 antibodies identified herein (e.g. SEQ ID NOS: 1 and2) other antibodies that bind to the same epitope can be readilyidentified. Thus, in one preferred embodiment, this invention providesan antibody that specifically binds to a c-erbB2 receptor epitope boundby F5 or C1. Particularly preferred antibodies of this type areinternalizing antibodies.

Because of the highly specific targeting to cells expression the c-erbB2receptor and the effective internalization of the bound molecules, theC1 and F5 antibodies of this invention are well suited for deliveringeffector molecules to or into a target cell (e.g. a cancer cell). Thus,in another preferred embodiment, this invention additionally providesfor chimeric molecules comprising the F5 or C1 antibodies (i.e.antibodies that bind to the epitope bound by F5 or C1) joined to aneffector molecule. The F5 or C1 antibody acts as a targeting moleculethat serves to specifically bind the chimeric molecule to cells bearingthe c-erbB-2 marker thereby delivering the effector molecule to thetarget cell.

An effector is a molecule or multimolecular structure (e.g. a liposome,a phage coat (capsid), or an intact phage) that is desired to bedelivered to the target cell (e.g. a tumor overexpressing c-erbB-2).Preferred effectors have a characteristic activity (e.g. drug delivery,cytotoxicity, fluorescence, radioactivity, etc.) Effector moleculesinclude, but are not limited to, cytotoxins, labels, radionuclides,ligands, antibodies, drugs, nucleic acids, hormones, growth factors,liposomes, and viral coat proteins that render a virus capable ofinfecting a c-erbB-2 expressing cell. Once delivered to the target, theeffector molecule exerts its characteristic activity.

For example, in one embodiment, where the effector molecule is acytotoxin, the chimeric molecule acts as a potent cell-killing agentspecifically targeting the cytotoxin to tumor cells bearing the c-erbB-2marker. Chimeric cytotoxins that specifically target tumor cells arewell known to those of skill in the art (see, for example, Pastan et al.(1992) Ann. Rev. Biochem., 61: 331-354).

In another embodiment, the chimeric molecule may be used for detectingthe presence or absence of tumor cells in vivo or in vitro or forlocalizing tumor cells in vivo. These methods involve providing achimeric molecule comprising an effector molecule, that is a detectablelabel. The C1 or F5 antibodies specifically bind the chimeric moleculeto tumor cells expressing the c-erbB-2 marker which are then marked bytheir association with the detectable label. Subsequent detection of thecell-associated label indicates the presence and/or mass and/or locationof a tumor cell.

In yet another embodiment, the effector molecule may be another specificbinding moiety including, but not limited to an antibody, an antigenbinding domain, a growth factor, or a ligand. The chimeric molecule willthen act as a highly specific bifunctional linker. This linker may actto bind and enhance the interaction between cells or cellular componentsto which the chimeric protein binds. Thus, for example, where the“effector” component is an anti-receptor antibody or antibody fragment,the C6 antibody component specifically binds c-erbB-2 bearing cancercells, while the effector component binds receptors (e.g., IL-2, IL-4,FcI, FcII and FcIII receptors) on the surface of immune cells. Thechimeric molecule may thus act to enhance and direct an immune responsetoward target cancer cells.

In still yet another embodiment the effector molecule may be apharmacological agent (e.g. a drug). Thus the C1 or F5 antibody may beconjugated to a drug such as vinblastine, vindesine, melphalan,N-acetylmelphalan, methotrexate, aminopterin, doxirubicin, daunorubicin,genistein (a tyrosine kinase inhibitor), an antisense molecule, andother pharmacological agents known to those of skill in the art, therebyspecifically targeting the pharmacological agent to tumor cellsexpressing c-erbB-2.

Alternatively, the F5 or C1 antibodies may be bound to a vehiclecontaining the therapeutic composition. Such vehicles include, but arenot limited to liposomes, micelles, various synthetic beads, and thelike.

One of skill in the art will appreciate that the chimeric molecules ofthe present invention optionally includes multiple targeting moieties(F5 and/or C1 antibodies) bound to a single effector or conversely,multiple effector molecules bound to a single targeting moiety. In stillother embodiment, the chimeric molecules include both multiple targetingmoieties and multiple effector molecules. Thus, for example, thisinvention provides for “dual targeted” cytotoxic chimeric molecules inwhich the F5 or C1 antibody is attached to a cytotoxic molecule whileanother molecule (e.g. an antibody, or another ligand) is attached tothe other terminus of the toxin. Such a dual-targeted cytotoxin mightcomprise, e.g. an F5 or C1 antibody substituted for domain Ia at theamino terminus of a PE and anti-TAC(Fv) inserted in domain III. Otherantibodies may also be suitable effector molecules.

As indicated above, preferred F5 and C1 antibodies of this invention areinternalizing antibodies. Often such internalizing antibodies have abiological activity even without the presence of an effector molecule.Many receptors (for example growth factor receptors) use internalizationas a way of modulating and regulating the effect of ligands. Forexample, ligand binding can result in signal transduction and receptorinternalization. The decrease in the number of receptors then causesdown regulation of the effect of additional ligand. The same occurs withantibodies that bind growth factor receptors (Hurwitz et al. (1995)Proc. Natl. Acad. Sci. USA. 92: 3353-3357). For example, “[g]rowthfactors act by binding to and activating the intrinsic catalyticactivity of their cell surface receptors, thereby initiating a signalingcascade leading to the cellular response. Growth factor/receptorcomplexes are not static residents of the cell surface membrane butundergo endocytic trafficking processes of internalization and sortingto recycling or degradation. Consequently, growth factors are depletedfrom the extracellular medium and their receptors undergodown-regulation. These trafficking processes, by virtue of theirinfluence on the kinetics of signaling growth factor/receptor complexes,are important modulators of cell behavioral responses” (Reddy et al.(1996) Nature Biotech. 14: 1696-1699)

In the ErbB2 system, one mechanism by which ErbB2 binding antibodiesinhibit growth is to cause receptor internalization and down regulation(Hurwitz et al. (1995) Proc. Natl. Acad. Sci. USA. 92: 3353-3357). Italso may be possible to turn an internalizing antibody that binds agrowth factor receptor into a growth inhibitor or stimulatory antibody.For example, the mitogenic properties of EGF have been increased bylowering the affinity of EGF for the EGF receptor. The lower affinityEGF causes receptor signaling, but reduced internalization and downregulation than wild type EGF (presumably from the lower affinity)(Reddy et al. (1996) Nature Biotech. 14: 1696-1699). Thus lowering theaffinity of a C1 or F5 internalizing antibody could turn it into agrowth factor. The F5 and C1 internalizing antibodies of this inventioncan provide lead compounds/drugs for both growth inhibition and growthstimulation.

II. Preparation/Synthesis of F5 and C1 Antibodies.

Using the sequence information provided herein, the explicitly listed F1and C1 antibodies can be routinely created either by de novo chemicalsynthesis or by recombinant DNA expression techniques. Similarlymodified F5 and C1 antibodies identified according to the methods aswell as the chimeric molecules of this invention can be synthesized denovo or recombinantly expressed (particularly where the chimericmolecule is a fusion protein.

A) Chemical Synthesis.

The F5 and C1 antibodies of this invention can be chemically synthesizedusing well known methods of peptide synthesis. Solid phase synthesis inwhich the C-terminal amino acid of the sequence is attached to aninsoluble support followed by sequential addition of the remaining aminoacids in the sequence one preferred method for the chemical synthesis ofC1 and F5 antibodies. Techniques for solid phase synthesis are describedby Barany and Merrifield, Solid Phase Peptide Synthesis; pp. 3-284 inThe Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A., Merrifield et al. (1963) J. Am. Chem. Soc.,85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis,2nd ed. Pierce Chem. Co., Rockford, Ill.

Typically F5 and C1 antibodies are chemically synthesized using anautomated peptide synthesizer such as an Eppendorf Synostat (Madison,Wis.) or a Milligen 9050 (Milford, Mass.), although manual methods ofcomposition peptide synthesis also can be used. In one embodiment,synthesis is on a polyethylene glycolpolystyrene (PEG-PS) graft resinand using N^(α)-Fmoc amino acid derivatives as described in U.S. Pat.No. 5,547,939. Other resins and synthesis chemistries (e.g. T-boc) canbe used.

B) Recombinant Expression.

In a preferred embodiment, the F5 or C1 antibodies of this invention areprepared using standard techniques well known to those of skill in theart in combination with the polypeptide and nucleic acid sequencesprovided herein. The polypeptide sequences may be used to determineappropriate nucleic acid sequences encoding the particular F5 or C1antibody disclosed thereby. The nucleic acid sequence may be optimizedto reflect particular codon “preferences” for various expression systemsaccording to standard methods well known to those of skill in the art.Alternatively, the nucleic acid sequences provided herein may also beused to express F5 or C1 antibodies.

Using the sequence information provided, the nucleic acids may besynthesized according to a number of standard methods known to those ofskill in the art. Oligonucleotide synthesis, is preferably carried outon commercially available solid phase oligonucleotide synthesis machines(Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12: 6159-6168) ormanually synthesized using the solid phase phosphoramidite triestermethod described by Beaucage et. al. (Beaucage et. al. (1981)Tetrahedron Letts. 22(20): 1859-1862).

Once a nucleic acid encoding a F5 or C1 antibody is synthesized it maybe amplified and/or cloned according to standard methods. Molecularcloning techniques to achieve these ends are known in the art. A widevariety of cloning and in vitro amplification methods suitable for theconstruction of recombinant nucleic acids, e.g., encoding F5 or C1antibody genes, are known to persons of skill. Examples of thesetechniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods ofproducing recombinant immunoglobulins are also known in the art. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Natl.Acad. Sci. USA 86: 10029-10033.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR) the ligase chain reaction (LCR), Q∃-replicase amplification andother RNA polymerase mediated techniques are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem. 35, 1826; Landegren et al., (1988) Science 241, 1077-1080;Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene4, 560; and Barringer et al. (1990) Gene 89, 117. Improved methods ofcloning in vitro amplified nucleic acids are described in Wallace etal., U.S. Pat. No. 5,426,039.

Once the nucleic acid for a F5 or C1 antibody is isolated and cloned,one may express the gene in a variety of recombinantly engineered cellsknown to those of skill in the art. Examples of such cells includebacteria, yeast, filamentous fungi, insect (especially employingbaculoviral vectors), and mammalian cells. It is expected that those ofskill in the art are knowledgeable in the numerous expression systemsavailable for expression of F5 or C1 antibodies.

In brief summary, the expression of natural or synthetic nucleic acidsencoding F5 or C1 antibodies will typically be achieved by operablylinking a nucleic acid encoding the antibody to a promoter (which iseither constitutive or inducible), and incorporating the construct intoan expression vector. The vectors can be suitable for replication and/orintegration in prokaryotes, eukaryotes, or both. Typical cloning vectorscontain transcription and translation terminators, initiation sequences,and promoters useful for regulation of the expression of the nucleicacid encoding the F5 or C1 antibody. The vectors optionally comprisegeneric expression cassettes containing at least one independentterminator sequence, sequences permitting replication of the cassette inboth eukaryotes and prokaryotes, i.e., shuttle vectors, and selectionmarkers for both prokaryotic and eukaryotic systems. See Sambrook,supra.

To obtain high levels of expression of a cloned nucleic acid it iscommon to construct expression plasmids which typically contain a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.Examples of regulatory regions suitable for this purpose in E. coli arethe promoter and operator region of the E. coli tryptophan biosyntheticpathway as described by Yanofsky (1984) J. Bacteriol., 158:1018-1024 andthe leftward promoter of phage lambda (P_(L)) as described by Herskowitzand Hagen (1980) Ann. Rev. Genet., 14: 399-445. The inclusion ofselection markers in DNA vectors transformed in E. coli is also useful.Examples of such markers include genes specifying resistance toampicillin, tetracycline, or chloramphenicol. See Sambrook for detailsconcerning selection markers, e.g., for use in E. coli.

Expression systems for expressing F5 or C1 antibodies are availableusing E. coli, Bacillus sp. (Palva et al. (1983) Gene 22:229-235;Mosbach, et al., Nature, 302:543-545) and Salmonella. E. coli systemsare preferred.

The F5 or C1 antibodies produced by prokaryotic cells may requireexposure to chaotropic agents for proper folding. During purificationfrom, e.g., E. coli, the expressed protein is optionally denatured andthen renatured. This is accomplished, e.g., by solubilizing thebacterially produced antibodies in a chaotropic agent such as guanidineHCl. The antibody is then renatured, either by slow dialysis or by gelfiltration. See, U.S. Pat. No. 4,511,503.

Methods of transfecting and expressing genes in mammalian cells areknown in the art. Transducing cells with nucleic acids can involve, forexample, incubating viral vectors containing F5 or C1 nucleic acids withcells within the host range of the vector. See, e.g., Methods inEnzymology, vol. 185, Academic Press, Inc., San Diego, Calif. (D. V.Goeddel, ed.) (1990) or M. Krieger, Gene Transfer and Expression—ALaboratory Manual, Stockton Press, New York, N.Y., (1990) and thereferences cited therein.

The culture of cells used in the present invention, including cell linesand cultured cells from tissue or blood samples is well known in theart. Freshney (Culture of Animal Cells, a Manual of Basic Technique,third edition Wiley-Liss, New York (1994)) and the references citedtherein provides a general guide to the culture of cells.

Techniques for using and manipulating antibodies are found in Coligan(1991) Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.)Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature256: 495-497.

In one preferred embodiment the F5 or C1 antibody gene is subcloned intoan expression vector, e.g., pUC119Sfi/NotHismyc (Schier et al. (1995)Immunotechnology. 1: 63-71). This results in the addition of ahexa-histidine tag at the C-terminal end of the scFv. A pHEN-1 vectorDNA containing the F5 or C1 scFv DNA is prepared by alkaline lysisminiprep, digested with NcoI and NotI, and the scFv DNA purified on a1.5% agarose gel. The F5 or C1 scFv DNA is ligated intopUC119Sfi1/Not1Hismyc digested with NcoI and NotI and the ligationmixture used to transform electrocompetent E. coli HB2151. Forexpression, 200 ml of 2×TY media containing 100:g/ml ampicillin and 0.1%glucose is inoculated with E. coli HB2151 harboring the F5 or C1 gene inpUC119Sfi1/Not1Hismyc. The culture is grown at 37° C. to an A₆₀₀ nm of0.8. Soluble scFv is expression induced by the addition of IPTG to afinal concentration of 1 mM, and the culture is grown at 30° C. in ashaker flask overnight.

The F5 or C1 antibodies may then be harvested from the periplasm usingthe following protocol: Cells are harvested by centrifugation at 4000 gfor 15 min, resuspended in 10 ml of ice cold 30 mM Tris-HCl pH 8.0, 1 mMEDTA, 20% sucrose, and incubated on ice for 20 minutes. The bacteria arethen pelleted by centrifugation at 6000 g for 15 min. and the“periplasmic fraction” cleared by centrifugation at 30,000 g for 20 min.The supernatant is then dialyzed overnight at 4° C. against 8 L of IMACloading buffer (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 20 mMimidazole) and then filtered through a 0.2 micron filter.

In a preferred embodiment, the F5 and/or C1 scFv is purified by IMAC.All steps are preferably performed at 4° C. A column containing 2 ml ofNi-NTA resin (Qiagen) is washed with 20 ml IMAC column wash buffer (50mM sodium phosphate pH 7.5, 500 mM NaCl, 250 mM imidazole) and 20 ml ofIMAC loading buffer. The periplasmic preparation is then loaded onto thecolumn and the column washed sequentially with 50 ml IMAC loading bufferand 50 ml IMAC washing buffer (50 mM sodium phosphate pH 7.5, 500 mMNaCl, 25 mM imidazole). Protein was eluted with 25 ml IMAC elutionbuffer (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 100 mM imidazole)and 4 ml fractions collected. The F5 and C1 antibody may be detected byabsorbance at 280 nm and scFv fraction eluted. To remove dimeric andaggregated scFv, samples can be concentrated to a volume <1 ml in aCentricon 10 (Amicon) and fractionated on a Superdex 75 column using arunning buffer of HBS (10 mM Hepes, 150 mM NaCl, pH 7.4).

The purity of the final preparation may be evaluated by assaying analiquot by SDS-PAGE. The protein bands can be detected by Coomassiestaining. The concentration can then be determinedspectrophotometrically, assuming that an A₂₈₀ nm of 1.0 corresponds toan scFv concentration of 0.7 mg/ml.

III. Modification of and/or Selection of Modified F5 and C1 Antibodies.

In a preferred embodiment, generation of new (different) F5 or C1antibodies involves generating an antibody (e.g. whole antibody,antibody fragment, or single chain antibody) and then screening theantibody to verify that it is an F5 or C1 antibody (i.e. that it bindsthe epitope bound by F5 or C1 and more preferably that it isinternalized). The antibodies to be screened can be randomly generatedby a variety of means, produced in vivo (e.g. by immunization of ananimal with a c-erbB2 epitope), or produced ex vivo, e.g. in a phage, orother, display library. The antibodies thus produced are then screenedfor c-erbB2 binding affinity and/or for specific binding to an F5 or C1epitope, and/or for internalization into a cell bearing the c-erbB2receptor or fragment(s) thereof.

A) Generation of F5- and C1-Derived Antibodies for Screening.

Alternatively the F5- and C1-derived antibodies are preferably obtainedby one of several strategies utilizing the F5 and C1 sequences providedherein. Such methods include, but are not limited to: 1) derivation bychain shuffling; 2) derivation by site directed mutagenesis of CDRs; 3)derivation by introducing multiple mutations into sequence by eithererror prone PCR, mutator strains of E. coli, or “DNA shuffling”. Thesederived antibodies include ‘library approaches’ where libraries ofmutant sequences based on F5 or C1 are created and binding function isthen selected for.

1) Generation of Page-Display Libraries.

The ability to express antibody fragments on the surface of viruses thatinfect bacteria (bacteriophage or phage) makes it possible to isolate asingle binding antibody fragment from a library of greater than 10¹⁰nonbinding clones. To express antibody fragments on the surface of phage(phage display), an antibody fragment gene is inserted into the geneencoding a phage surface protein (pIII) and the antibody fragment-pIIIfusion protein is displayed on the phage surface (McCafferty et al.(1990) Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res.19: 4133-4137). Since the antibody fragments on the surface of the phageare functional, phage bearing antigen binding antibody fragments can beseparated from non-binding phage by antigen affinity chromatography(McCafferty et al. (1990) Nature, 348: 552-554). Depending on theaffinity of the antibody fragment, enrichment factors of 20fold-1,000,000 fold are obtained for a single round of affinityselection. By infecting bacteria with the eluted phage, however, morephage can be grown and subjected to another round of selection. In thisway, an enrichment of 1000 fold in one round can become 1,000,000 foldin two rounds of selection (McCafferty et al. (1990) Nature, 348:552-554). Thus even when enrichments are low (Marks et al. (1991) J.Mol. Biol. 222: 581-597), multiple rounds of affinity selection can leadto the isolation of rare phage. Since selection of the phage antibodylibrary on antigen results in enrichment, the majority of clones bindantigen after four rounds of selection. Thus only a relatively smallnumber of clones (several hundred) need to be analyzed for binding toantigen. It is noted that in certain preferred embodiments, polyvalentphage display systems are preferred as indicated in the Examples.

In a preferred embodiment, analysis for binding is simplified byincluding an amber codon between the antibody fragment gene and geneIII. The amber codon makes it possible to easily switch betweendisplayed and soluble (native) antibody fragment simply by changing thehost bacterial strain (Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137).

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581-597). In one embodiment natural V_(H)and V_(L) repertoires present in human peripheral blood lymphocytes wereisolated from unimmunized donors by PCR. The V-gene repertoires werespliced together at random using PCR to create a scFv gene repertoirewhich was cloned into a phage vector to create a library of 30 millionphage antibodies (Id.). From this single “naive” phage antibody library,binding antibody fragments have been isolated against more than 17different antigens, including haptens, polysaccharides and proteins(Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993).Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies havebeen produced against self proteins, including human thyroglobulin,immunoglobulin, tumor necrosis factor and CEA (Griffiths et al. (1993)EMBO J. 12: 725-734). It is also possible to isolate antibodies againstcell surface antigens by selecting directly on intact cells. Forexample, antibody fragments against four different erythrocyte cellsurface antigens were produced by selecting directly on erythrocytes(Marks et al. (1993). Bio/Technology. 10: 779-783). Antibodies wereproduced against blood group antigens with surface densities as low as5,000 sites/cell. The antibody fragments were highly specific to theantigen used for selection, and were functional in agglutination andimmunofluorescence assays. Antibodies against the lower density antigenswere produced by first selecting the phage antibody library on a highlyrelated cell type which lacked the antigen of interest. This negativeselection removed binders against the higher density antigens andsubsequent selection of the depleted phage antibody library on cellsexpressing the antigen of interest resulted in isolation of antibodiesagainst that antigen. With a library of this size and diversity, atleast one to several binders can be isolated against a protein antigen70% of the time. The antibody fragments are highly specific for theantigen used for selection and have affinities in the 1:M to 100 nMrange (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Griffiths et al.(1993) EMBO J. 12: 725-734). Larger phage antibody libraries result inthe isolation of more antibodies of higher binding affinity to a greaterproportion of antigens.

The creation of a suitable large phage display antibody library isdescribed in detail in the examples provided herein.

2) Phage Display can be Used to Increase Antibody Affinity.

To create higher affinity antibodies, mutant antibody gene repertories,based on the sequence of a binding F5 or C1 antibody (e.g., based on thescFv F5 or C1 antibodies described herein), are created and expressed onthe surface of phage. Higher affinity scFvs are selected on antigen asdescribed above, in the Examples, and in Schier et al. (1996) j. Mol.Biol., 263: 551-567.

Higher affinity scFvs are selected by affinity chromatography on antigenas described above. One approach to creating mutant scFv generepertoires has been to replace the original V_(H) or V_(L) gene with arepertoire of V-genes to create new partners (chain shuffling) (Clacksonet al. (1991) Nature. 352: 624-628). Using chain shuffling and phagedisplay, the affinity of a human scFv antibody fragment which bound thehapten phenyloxazolone (phOx) was increased from 300 nM to 1 nM (300fold) (Marks et al. (1992) Bio/Technology 10: 779-783).

Thus, for example, to alter the affinity of F5 or C1 antibodies, amutant scFv gene repertoire can be created containing the V_(H) gene ofF5 or C1 and a human V_(L) gene repertoire (light chain shuffling). ThescFv gene repertoire can be cloned into the phage display vector pHEN-1(Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133-4137) and aftertransformation a library of transformants is obtained. Phage areprepared and concentrated as described in the Examples.

Similarly, for heavy chain shuffling, the F5 or C1 V_(H) CDR1 and/orCDR2, and/or CDR3 and light chain are cloned into a vector containing ahuman V_(H) gene repertoire to create a phage antibody librarytransformants. For detailed descriptions of chain shuffling to increaseantibody affinity see Schier et al. (1996) J. Mol. Biol., 255: 28-43,1996.

C1 and F5 selections can be performed by incubating the phage withbiotinylated c-erbB-2 in solution. The antigen concentration isdecreased each round, reaching a concentration less than the desiredK_(d) by the final rounds of selection. This results in the selection ofphage on the basis of affinity (Hawkins et al. (1992) J. Mol. Biol. 226:889-896).

3) Site-Directed Mutagenesis to Improve Binding Affinity.

The majority of antigen contacting amino acid side chains are located inthe complementarity determining regions (CDRs), three in the V_(H)(CDR1, CDR2, and CDR3) and three in the V_(L) (CDR1, CDR2, and CDR3)(Chothia et al. (1987) J. Mol. Biol., 196: 901-917; Chothia et al.(1986) Science, 233: 755-8; Nhan et al. (1991) J. Mol. Biol., 217:133-151). These residues contribute the majority of binding energeticsresponsible for antibody affinity for antigen. In other molecules,mutating amino acids which contact ligand has been shown to be aneffective means of increasing the affinity of one protein molecule forits binding partner (Lowman et al. (1993) J. Mol. Biol., 234: 564-578;Wells (1990) Biochemistry, 29: 8509-8516). Site-directed mutagenesis ofCDRs and screening against c-erbB-2 may be used to generate C6antibodies having improved binding affinity and/or internalization of F5and C1 antibodies.

4) CDR Randomization to Produce Higher Affinity Human scFv.

In an extension of simple site-directed mutagenesis, mutant antibodylibraries can be created where partial or entire CDRs are randomized(V_(L) CDR1 and CDR2 and V_(H) CDR1, CDR2 and CDR3). In one embodiment,each CDR is randomized in a separate library, using F5 or C1 as atemplate. The CDR sequences of the highest affinity mutants from eachCDR library are combined to obtain an additive increase in affinity. Asimilar approach has been used to increase the affinity of human growthhormone (hGH) for the growth hormone receptor over 1500 fold from3.4×10⁻¹⁰ to 9.0×10⁻¹³ M (Lowman et al. (1993) J. Mol. Biol., 234:564-578).

V_(H) CDR3 occupies the center of the binding pocket, and thus mutationsin this region are likely to result in an increase in affinity (Clacksonet al. (1995) Science, 267: 383-386). In one embodiment, four V_(H) CDR3residues are randomized at a time using the nucleotides NNS (see, e.g.,Schier et al. (1996) Gene, 169: 147-155; Schier and Marks (1996) HumanAntibodies and Hybridomas. 7: 97-105, 1996; and Schier et al. (1996) J.Mol. Biol. 263: 551-567, 1996).

To create the library, an oligonucleotide is synthesized which annealsto the F5 or C1 V_(H) framework 3 and encodes V_(H) CDR3 and a portionof framework 4. At the four positions to be randomized, the sequence NNSis used, where N=any of the 4 nucleotides, and S═C or T. Theoligonucleotides are used to amplify the F5 or C1 V_(H) genes using PCR,creating a mutant F5 or C1 V_(H) gene repertoire. PCR is used to splicethe V_(H) gene repertoire with the C6NIL3-Bl light chain gene, and theresulting scFv gene repertoire cloned into the phage display vectorpHEN-1. Ligated vector DNA is used to transform electrocompetent E. colito produce a phage antibody library of >1.0×10⁷ clones (Id.).

To select higher affinity mutant scFv, each round of selection of thephage antibody libraries is conducted on decreasing amounts ofbiotinylated c-erbB-2, as described above. Typically, 96 clones from thethird and fourth round of selection are screened for binding to c-erbB-2by ELISA on 96 well plates. scFv from twenty to forty ELISA positiveclones are expressed in 10 ml cultures, the periplasm harvested, and thescFv k_(off) determined by BIAcore. Clones with the slowest k_(off) aresequenced, and each unique scFv subcloned, e.g., into pUC119Sf-NotmycHis. scFv is expressed in 1 L cultures, and purified asdescribed supra. Affinities of purified scFv are determined by BIAcore.

5) Creation of Homodimers.

To create F5 or C1 (scFv′)₂ antibodies, two F5 or two C1 scFvs arejoined, either through a linker (e.g., a carbon linker, a peptide, etc.)or through a disulfide bond between, for example, two cysteins. Thus,for example, to create disulfide linked F5 scFv, a cysteine residue isintroduced by site directed mutagenesis between the myc tag andhexahistidine tag at the carboxy-terminus of F5. Introduction of thecorrect sequence can be verified by DNA sequencing. If the construct isin pUC119, the pelB leader directs expressed scFv to the periplasm andcloning sites (Nco1 and Not1) exist to introduce F5 or C1 mutant scFv.The expressed scFv has the myc tag at the C-terminus, followed by 2glycines, a cysteine, and then 6 histidines to facilitate purificationby IMAC. After disulfide bond formation between the two cysteineresidues, the two scFv are separated from each other by about 26 aminoacids (two 11 amino acid myc tags and 4 glycines).

An scFv can be expressed from this construct, purified by IMAC, andanalyzed by gel filtration. To produce (scFv′)₂ dimers, the cysteine isreduced by incubation with 1 mM ∃-mercaptoethanol, and half of the scFvblocked by the addition of DTNB. Blocked and unblocked scFvs areincubated together to form (scFv′)₂ and the resulting material can beanalyzed by gel filtration. The affinity of the F5 and C1 scFv′ monomersand the F5 and C1 (scFv′)₂ dimers is determined by BIAcore.

In a particularly preferred embodiment, the (scFv′)₂ dimer is created byjoining the scFv′ fragments through a linker, more preferably through apeptide linker. This can be accomplished by a wide variety of means wellknown to those of skill in the art. For example, one preferred approachis described by Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448 (see also WO 94/13804).

B) Recognizing/Selecting F5 or C1 Antibodies

As indicated above, in one embodiment, this invention provides twoanti-c-erbB-2 antibodies that are effectively internalized. Theseantibodies, designated herein as F5 (SEQ ID NO: 1) and C1 (SEQ ID NO:2), specifically bind to a previously unrecognized epitope on theexternal domain of c-erB-2, and thereby characterize that epitope. Usingthe information provided provided, other F5 and C1 antibodies that bindthe same epitope can be routinely prepared.

Regardless of the method used to generate new C1 or F5 antibodies (e.g.animal immunization with c-erbB2, domain shuffling in a single-chainantibody library, etc.) to verify that the antibody produced is apreferred F5 or C1 antibody, the putative F5 or C1 antibody ispreferably screened for 1) binding affinity for c-erbB2; and/or 2)specific binding of the F5 and/or C1 epitope; and 3) internalization.

1) Measurement of Antibody/Polypeptide Binding Affinity.

As explained above, selection for increased avidity involves measuringthe affinity of the antibody (e.g. a modified F5 or C1) for the targetantigen (e.g., c-erbB-2). Methods of making such measurements aredescribed in detail in copending application U.S. Ser. No. 08/665,202.Briefly, for example, the K_(d) of F5, C1, or an F5- or C1-derivedantibody the kinetics of binding to c-erbB-2 are determined in aBIAcore, a biosensor based on surface plasmon resonance. For thistechnique, antigen is coupled to a derivatized sensor chip capable ofdetecting changes in mass. When antibody is passed over the sensor chip,antibody binds to the antigen resulting in an increase in mass that isquantifiable. Measurement of the rate of association as a function ofantibody concentration can be used to calculate the association rateconstant (k_(on)). After the association phase, buffer is passed overthe chip and the rate of dissociation of antibody (k_(off)) determined.K_(on) is typically measured in the range 1.0×10² to 5.0×10⁶ and k_(off)in the range 1.0×10⁻¹ to 1.0×10⁻⁶. The equilibrium constant K_(d) isoften calculated as k_(off)/k_(on) and thus is typically measured in therange 10⁻⁵ to 10⁻¹². Affinities measured in this manner correlate wellwith affinities measured in solution by fluorescence quench titration.

Preferred F5 and C1 antibodies bind to the c-erbB-2 F5 and C1 epitope oncells with a binding affinity of at least 10⁻⁵ M, more preferably with abinding affinity of at least 1 μM, and most preferably with a bindingaffinity of at least 100 nM, 10 nM, or even 1 nM.

2) Identification of Antibodies that Bind the Epitope Bound by F5 and/orC1

The F5 and C1 antibodies (e.g., antibodies that bind the same epitopebound by C1 and F5) can be identified (selected) from serum derived froman appropriately immunized animal, or from an ex vivo system (e.g., arandom antibody library) by selection for antibodies that compete withC1 or F5 for binding to a c-erbB2 epitope. The F5 and C1 derivedantibodies are recognized by their cross-reactivity with either 1) thec-erbB-2 epitope recognized by F5 and C1; and/or 2) an anti F5 or C1idiotypic antibody. For targeting and internalization, high affinity F5and C1 antibodies are preferred, however, if it is desired to convert anantagonist to an agonist it is expected that low affinity F5 and C1antibodies may be preferred. Moreover, for many applications rapid andefficient internalization is more important than affinity and a loweraffinity antibody that is rapidly internalized may be preferred to ahigher affinity antibody that is not internalized as quickly.

a) Cross-Reactivity with Anti-Idiotypic Antibodies.

The idiotype represents the highly variable antigen-binding site of anantibody and is itself immunogenic. During the generation of anantibody-mediated immune response, an individual will develop antibodiesto the antigen as well as anti-idiotype antibodies, whose immunogenicbinding site (idiotype) mimics the antigen.

F5- and C1-derived antibodies can then be recognized by their ability tospecifically bind to F5 and C1-anti-idiotypic antibodies, i.e., F5- andC1-derived antibodies are preferably cross-reactive with F5 and C1 withF5 or C1 anti-idiotypic antibodies.

Anti-idiotypic antibodies can be raised against the variable regions ofF5 or C1 using standard methods well known to those of skill in the art.Briefly, anti-idiotype antibodies can be made by injecting antibodies ofthis invention (e.g. F5 or C1 antibodies or fragments thereof (e.g.,CDRs)) into an animal thereby eliciting antiserum against variousantigenic determinants on the antibody, including determinants in theidiotypic region.

Methods for the production of anti-analyte antibodies are well known inthe art. Large molecular weight antigens (greater than approx. 5000Daltons) can be injected directly into animals, whereas small molecularweight compounds (less than approx. 5000 Daltons) are preferably coupledto a high molecular weight immunogenic carrier, usually a protein, torender them immunogenic. The antibodies produced in response toimmunization can be utilized as serum, ascites fluid, an immunoglobulin(Ig) fraction, an IgG fraction, or as affinity-purified monospecificmaterial.

Polyclonal anti-idiotype antibodies can be prepared by immunizing ananimal with the antibodies of this invention prepared as describedabove. In general, it is desirable to immunize an animal which isspecies and allotype-matched with the animal from which the antibody(e.g. phage-display library) was derived. This minimizes the productionof antibodies directed against non-idiotypic determinants. The antiserumso obtained is then usually absorbed extensively against normal serumfrom the same species from which the phage-display library was derived,thereby eliminating antibodies directed against non-idiotypicdeterminants. Absorption can be accomplished by passing antiserum over agel formed by crosslinking normal (nonimmune) serum proteins withglutaraldehyde. Antibodies with anti-idiotypic specificity will passdirectly through the gel, while those having specificity fornon-idiotypic determinants will bind to the gel. Immobilizing nonimmuneserum proteins on an insoluble polysaccharide support (e.g., sepharose)also provides a suitable matrix for absorption.

Monoclonal anti-idiotype antibodies can be produced using the method ofKohler et al. (1975) Nature 256: 495. In particular, monoclonalanti-idiotype antibodies can be prepared using hybridoma technologywhich comprises fusing (1) spleen cells from a mouse immunized with theantigen or hapten-carrier conjugate of interest (i.e., the antibodies orthis invention or subsequences thereof) to (2) a mouse myeloma cell linewhich has been selected for resistance to a drug (e.g., 8-azaguanine).In general, it is desirable to use a myeloma cell line that does notsecrete an immunoglobulin. Several such lines are known in the art. Apreferred cell line is P3X63Ag8.653. This cell line is on deposit at theAmerican Type Culture Collection as CRL-1580.

Fusion can be carried out in the presence of polyethylene glycolaccording to established methods (see, e.g., Monoclonal Antibodies, R.Kennett, J. McKearn & K. Bechtol, eds. N.Y., Plenum Press, 1980, andCurrent Topics in Microbiology & Immunology, Vol. 81, F. Melchers, M.Potter & N. L. Warner, eds., N.Y., Springer-Verlag, 1978). The resultantmixture of fused and unfused cells is plated out inhypoxanthine-aminopterin-thymidine (HAT) selective medium. Under theseconditions, only hybrid cells will grow.

When sufficient cell growth has occurred, (typically 10-14 dayspost-fusion), the culture medium is harvested and screened for thepresence of monoclonal idiotypic, anti-analyte antibody by any one of anumber of methods which include solid phase RIA and enzyme-linkedimmunosorbent assay. Cells from culture wells containing antibody of thedesired specificity are then expanded and recloned. Cells from thosecultures which remain positive for the antibody of interest are thenusually passed as ascites tumors in susceptible, histocompatible,pristane-primed mice.

Ascites fluid is harvested by tapping the peritoneal cavity, retestedfor antibody, and purified as described above. If a nonsecreting myelomaline is used in the fusion, affinity purification of the monoclonalantibody is not usually necessary since the antibody is alreadyhomogeneous with respect to its antigen-binding characteristics. Allthat is necessary is to isolate it from contaminating proteins inascites, i.e., to produce an immunoglobulin fraction.

Alternatively, the hybrid cell lines of interest can be grown inserum-free tissue culture and the antibody harvested from the culturemedium. In general, this is a less desirable method of obtaining largequantities of antibody because the yield is low. It is also possible topass the cells intravenously in mice and to harvest the antibody fromserum. This method is generally not preferred because of the smallquantity of serum which can be obtained per bleed and because of theneed for extensive purification from other serum components. However,some hybridomas will not grow as ascites tumors and therefore one ofthese alternative methods of obtaining antibody must be used.

b) Cross-Reactivity with F5 or C1.

Instead of the anti-idiotypic antibody, putative F5- and C1-derivedantibodies can be identified by cross-reactivity with F5 and C1,respectively, against the c-erbB-2 F5 and C1 epitopes.

This can be ascertained by providing cells expressing native orrecombinant c-erbB-2 or by providing the isolated c-erbB-2 attached to asolid support. Competition between the putative F5 (or C1) and the F5 orC1 of SEQ ID NOS: 1 and 2, respectively, in an epitope-mapping formatestablishes that the antibodies are competing for the same epitope. Theputative antibodies are then screened as described below.

c) Cross-Reactivity Measurements.

Immunoassays in the competitive binding format are preferably used forcrossreactivity determinations. For example, the F5 or C1 epitope oranti-idiotypic antibody is immobilized to a solid support. The putativeF5-derived or C1-derived antibodies (e.g. generated by selection from aphage-display library) added to the assay compete with F5 or C1antibodies of SEQ ID NOS 1 and 2, respectively binding to theimmobilized epitope or anti-idiotypic antibody. The ability of theputative F5-derived or C1-derived antibodies to compete with the bindingof the F5 or C1 antibodies (SEQ ID NOS: 1 and 2) to the immobilizedprotein are compared. The percent crossreactivity above proteins iscalculated, using standard calculations.

If the putative F5-derived or C1-derived antibody competes with F5 or C1and has a binding affinity comparable to or greater than F5 or C1 withthe same target then the putative F5-derived or C1-derived antibody isregarded as an F5 or C1 (derived) antibody.

3) Measurement of C1 and F5 Antibody Internalization.

In one embodiment, this invention provides methods for identifyinginternalizing F5 or C1 antibodies. The methods involve contacting a“target” cell with one or more putative F5 or C1 antibodies (e.g.members of a phage display library). After a suitable incubation period,the cells are washed to remove externally bound phage (library members)and then internalized phage are released from the cells by cell lysis.The internalized phage in the cell lysate can be recovered and expandedby using the lysate containing internalized phage to infect a bacterialhost. Growth of infected bacteria leads to expansion of the phage thatcan be used for a subsequent round of selection. Each round of selectionenriches for phage that are more efficiently internalized, more specificfor the target cell or have improved binding characteristics.

The phage display library is preferably contacted with a subtractivecell line (i.e. a subtractive cell line is added to the target cells andculture media) to remove members of the phage display library that arenot specific to the “target” cell(s). The subtractive cell line ispreferably added under conditions in which members of the phage displaylibrary are not internalized (e.g., at a temperature of about 4° C. toabout 20° C., more preferably at a temperature of about 4° C.) so thatnon-specific binding members of the library are not internalized(sequestered) before they can be subtracted out by the subtractive cellline.

After subtracting out non-specific binding antibodies, the “target”cells are washed to remove the subtractive cell line and to removenon-specifically or weakly-bound phage.”

The target cells are then cultured under conditions where it is possiblefor internalization to occur (e.g. at a temperature of about 35° C. toabout 39° C., more preferably at a temperature of about 37° C.). Theduration of the internalization culture period will determine theinternalization speed of the antibodies (phage display members) forwhich selection takes place. With shorter internalization periods morerapid internalizing antibodies are selected while with longerinternalization periods slower internalizing antibodies are selected.The internalization period is preferably less than about 120 minutes,more preferably less than about 60 minutes, and most preferably lessthan about 30 minutes or even less than about 20 minutes.

It is noted that during the internalization period the target cells aregrown under conditions in which internalization can occur. For a numberof cell lines, this involves culturing the cells adherently on cultureplates.

After internalization has been allowed to occur the target cells arewashed to remove non-internalized (e.g. surface-bound phage).

The cells can then be moved to clean media. In a preferred embodiment,where the cells are adherent, they cells are trypsinized to free thecells from the extracellular matrix which may contain phage antibodiesthat bind the extracellular matrix. Freeing the cells into solutionpermits more through washing and moving of the cells to a new cultureflask will leave behind any phage that may have stuck to the tissueculture dish.

The cells can then be washed with a large volume of PBS and lysed torelease the internalized phage which can then be expanded e.g. used toinfect E. coli to produce phage for the next round of selection. It isnoted that there is no need to actually visualize the internalizedphage. Simple cell lysis and expansion of the formerly internalizedphage is sufficient for recovering internalizing phage display members.Methods of selecting for internalizing phage library members are alsodescribed in related application U.S. Ser. No. 60/082,953 and in theExamples provided herein.

IV. F5 and C1 Epitopes.

In another embodiment, this invention provides for the epitope (s0specifically recognized and bound by the F5 and C1 antibodies of thisinvention. This internalizing epitope is characterized by the ability tobe specifically bound by F5 and C1 respectively. Thus, the F5 epitope isa region of c-erbB2 that specifically binds F5 (SEQ ID NO: 1) while theC1 epitope is a region of c-erbB2 that specifically binds C1 (SEQ ID NO:1). It is believed that F5 and C1 both bind to the same c-erbB2 epitope.

The F5 and C1 epitopes can identified by epitope mapping using standardtechniques (see, e.g., Geysen et al (1987) J. Immunol. Meth. 102,259-274). This technique involves the synthesis of overlapping c-erbB-2peptides. The synthesized peptides are then screened against F5 and C1respectively, and the characteristic F5 and C1 epitopes can beidentified by binding specificity and affinity.

The peptides for F5 and C1 epitope mapping can be conveniently preparedusing “Multipin” peptide synthesis techniques (see, e.g., Geysen et al(1987) Science, 235:1184-1190). Using the known sequence of c-erbB-2(see, e.g, SWISS-PROT: P04626 or Coussens et al. (1985) Science, 230:1132-1139), overlapping c-erbB-2 polypeptide sequences can besynthesized individually in a sequential manner on plastic pins in anarray of one or more 96-well microtest plate(s).

The procedure for epitope mapping using this multipin peptide system isdescribed in U.S. Pat. No. 5,739,306. Briefly, the pins are firsttreated with a pre-coat buffer containing 2% bovine serum albumin and0.1% Tween 20 in PBS for 1 hour at room temperature. Then the pins arethen inserted into the individual wells of 96-well microtest platecontaining antibody F5 or C1 in the pre-coat buffer at 2 mu g/ml. Theincubation is for 1 hour at room temperature. The pins are washed inPBST (3 rinses for every 10 minutes), and then incubated in the wells ofa 96-well microtest plate containing 100 mu 1 of HRP-conjugated goatanti-mouse IgG (Fc) (Jackson ImmunoResearch Laboratories) at a 1:4,000dilution for 1 hour at room temperature. After the pins are washed asbefore, the pins were put into wells containing peroxidase substratesolution of diammonium 2,2′-azino-bis[3-ethylbenzthiazoline-b-sulfonate](ABTS) and H₂O₂ (Kirkegaard & Perry Laboratories Inc., Gaithersburg,Md.) for 30 minutes at room temperature for color reaction. The plate isread at 405 nm by a plate reader (e.g., BioTek ELISA plate reader)against a background absorption wavelength of 492 nm. Wells showingcolor development indicated reactivity of the c-erbB-2 derived peptidesin such wells with F5 or C1.

V. Preparation of Chimeric Molecules.

In another embodiment this invention provides for chimeric moleculescomprising an F5 or C1 antibody attached to an effector molecule. Asexplained above, the effector molecule component of the chimericmolecules of this invention may be any molecule whose activity it isdesired to deliver to cells that express c-erbB-2. Suitable effectormolecules include cytotoxins such as PE, Ricin, Abrin or DT,radionuclides, ligands such as growth factors, antibodies, detectablelabels such as fluorescent or radioactive labels, and therapeuticcompositions such directly fused or conjugated drugs, “capsules” (e.g.liposomes, synthetic polymer capsules, virus capsids, live virus)containing various drugs or other agents (e.g. nucleic acids).

An effector molecule typically has a characteristic activity that isdesired to be delivered to the target cell (e.g. a tumor overexpressingc-erbB-2). Effector molecules include cytotoxins, labels, radionuclides,ligands, antibodies, drugs, liposomes, viruses or viral capsidscontaining nucleic acids ((e.g. DNA, RNA, antisense molecules, peptidenucleic acids, etc.), and viral coat proteins that render the viruscapable of infecting a c-erbB-2 expressing cell. Once delivered to thetarget, the effector molecule exerts its characteristic activity.

For example, in one embodiment, where the effector molecule is acytotoxin, the chimeric molecule acts as a potent cell-killing agentspecifically targeting the cytotoxin to tumor cells bearing the c-erbB-2marker. Chimeric cytotoxins that specifically target tumor cells arewell known to those of skill in the art (see, e.g., Pastan et al. (1992)Ann. Rev. Biochem., 61: 331-354).

In another embodiment, the chimeric molecule may be used for detectingthe presence or absence of tumor cells in vivo or in vitro, and/or forquantifying tumor cells in vivo or in vitro, and/or for localizing tumorcells in vivo. These methods involve providing a chimeric moleculecomprising an effector molecule, that is a detectable label attached tothe F5 or C1 antibody. The F5 or C1 antibodies specifically bind thechimeric molecule to tumor cells expressing the c-erbB-2 marker whichare then marked by their association with the detectable label.Subsequent detection of the cell-associated label indicates the presenceand/or location of a tumor cell.

In yet another embodiment, the effector molecule may be another specificbinding moiety including, but not limited to an antibody, an antigenbinding domain, a growth factor, or a ligand. The chimeric molecule willthen act as a highly specific bifunctional linker. This linker may actto bind and enhance the interaction between cells or cellular componentsto which the chimeric protein binds. Thus, for example, where the“effector” component is an anti-receptor antibody or antibody fragment,the F5 or C1 antibody component specifically binds c-erbB-2 bearingcancer cells, while the effector component binds receptors (e.g., IL-2,IL-4, FcI, FcII and FcIII receptors) on the surface of immune cells. Thechimeric molecule may thus act to enhance and direct an immune responsetoward target cancer cells.

In still yet another embodiment the effector molecule may be apharmacological agent (e.g. a drug) or a vehicle containing apharmacological agent. This is particularly suitable where it is merelydesired to invoke a non-lethal biological response. Thus the F5 or C1antibody receptor may be conjugated to a drug such as vinblastine,vindesine, melphalan, N-Acetylmelphalan, methotrexate, aminopterin,doxirubicin, daunorubicin, genistein (a tyrosine kinase inhibitor), anantisense molecule, and other pharmacological agents known to those ofskill in the art, thereby specifically targeting the pharmacologicalagent to tumor cells expressing c-erbB-2.

Alternatively, the F5 or C1 antibody may be bound to a vehiclecontaining the therapeutic composition. Such vehicles include, but arenot limited to liposomes, micelles, various synthetic beads or polymercapsules, virus capsids, live virus, and the like.

One of skill in the art will appreciate that the chimeric molecules ofthe present invention optionally includes multiple targeting moietiesbound to a single effector or conversely, multiple effector moleculesbound to a single targeting moiety. In still other embodiment, thechimeric molecules includes both multiple targeting moieties andmultiple effector molecules. Thus, for example, this invention providesfor “dual targeted” cytotoxic chimeric molecules in which the F5 or C1antibody is attached to a cytotoxic molecule while another molecule(e.g. an antibody, or another ligand) is attached to the other terminusof the toxin. Such a dual-targeted cytotoxin might comprise, e.g. an F5or C1 antibody substituted for domain Ia at the amino terminus of a PEand anti-TAC(Fv) inserted in domain III. Other antibodies may also besuitable effector molecules.

A) Cytotoxins.

Particularly preferred cytotoxins include Pseudomonas exotoxins,Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin and Dipthteriatoxin, in particular, are frequently used in chimeric cytotoxins.

1) Pseudomonas exotoxin (PE).

Pseudomonas exotoxin A (PE) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells through the inactivationof elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation(catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD ontoEF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity. See Siegall et al. (1989) J.Biol. Chem. 264: 14256-14261.

For maximum cytotoxic properties of a preferred PE molecule, severalmodifications to the molecule are recommended. An appropriate carboxylterminal sequence to the recombinant molecule is preferred totranslocate the molecule into the cytosol of target cells. Amino acidsequences which have been found to be effective include, REDLK (as innative PE), REDL, RDEL, or KDEL, repeats of those, or other sequencesthat function to maintain or recycle proteins into the endoplasmicreticulum, referred to here as “endoplasmic retention sequences”. See,for example, Chaudhary et al. (1991) Proc. Natl. Acad. Sci. USA87:308-312 and Seetharam et al (1991) J. Biol. Chem. 266: 17376-17381.

The targeting molecule can be inserted in replacement for domain Ia. Asimilar insertion has been accomplished in what is known as the TGF-PE40molecule (also referred to as TP40) described in Heimbrook et al. (1990)Proc. Natl. Acad. Sci., USA, 87: 4697-4701. See also, Debinski et al.(1994) Bioconj. Chem., 5: 40, for other PE variants).

The PE molecules can be fused to the F5 or C1 antibody by recombinantmeans. The genes encoding protein chains may be cloned in cDNA or ingenomic form by any cloning procedure known to those skilled in the art.See for example Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, (1989). Methods of cloning genes encodingPE fused to various ligands are well known to those of skill in the art.See, for example, Siegall et al. (1989) FASEB J., 3: 2647-2652;Chaudhary et al. (1997) Proc. Natl. Acad. Sci. USA, 84: 4538-4542.

Those skilled in the art will realize that additional modifications,deletions, insertions and the like may be made to the chimeric moleculesof the present invention or to the nucleic acid sequences encoding theF5 or C1 chimeric molecules. Especially, deletions or changes may bemade in PE or in a linker connecting an antibody gene to PE, in order toincrease cytotoxicity of the fusion protein toward target cells or todecrease nonspecific cytotoxicity toward cells without antigen for theantibody. All such constructions may be made by methods of geneticengineering well known to those skilled in the art (see, generally,Sambrook et al., supra) and may produce proteins that have differingproperties of affinity, specificity, stability and toxicity that makethem particularly suitable for various clinical or biologicalapplications.

2) Diphtheria Toxin (DT).

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 (EF-2) thereby inhibiting protein synthesis.Diphtheria toxin, however, is divided into two chains, A and B, linkedby a disulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida etal., (1972) Science, 175: 901-903; Uchida et al. (1973) J. Biol. Chem.,248: 3838-3844).

The targeting molecule-Diphtheria toxin fusion proteins of thisinvention may have the native receptor-binding domain removed bytruncation of the Diphtheria toxin B chain. DT388, a DT in which thecarboxyl terminal sequence beginning at residue 389 is removed isillustrated in Chaudhary, et al. (1991) Bioch. Biophys. Res. Comm., 180:545-551.

Like the PE chimeric cytotoxins, the DT molecules may be chemicallyconjugated to the F5 or C1 antibody, but may also be prepared as fusionproteins by recombinant means. The genes encoding protein chains may becloned in cDNA or in genomic form by any cloning procedure known tothose skilled in the art. Methods of cloning genes encoding DT fused tovarious ligands are also well known to those of skill in the art. See,for example, Williams et al. (1990) J. Biol. Chem. 265: 11885-11889which describes the expression of growth-factor-DT fusion proteins.

The term “Diphtheria toxin” (DT) as used herein refers to full lengthnative DT or to a DT that has been modified. Modifications typicallyinclude removal of the targeting domain in the B chain and, morespecifically, involve truncations of the carboxyl region of the B chain.

B) Detectable Labels.

The term “detectable label” refers to any material having a detectablephysical or chemical property. Such detectable labels have beenwell-developed in the field of immunoassays and, in general, any labeluseful in such methods can be applied to the present invention. Thus, alabel is any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texasred, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), fluorescent proteins (e.g. green fluorescent protein (GFP), bluefluorescent protein (BFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), luciferase), enzymes (e.g., LacZ, CAT, horseradish peroxidase, alkaline phosphatase and others, commonly used asdetectable enzymes, either as marker gene products or in an ELISA), andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

It will be recognized that fluorescent labels are not to be limited tosingle species organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe—CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots(zinc sulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016-2018).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. Non radioactivelabels are often attached by indirect means. Generally, a ligandmolecule (e.g., biotin) is covalently bound to the molecule. The ligandthen binds to an anti-ligand (e.g., streptavidin) molecule which iseither inherently detectable or covalently bound to a signal system,such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody. Themolecules can also be conjugated directly or thorough a linker to signalgenerating compounds.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

C) Ligands.

As explained above, the effector molecule may also be a ligand or anantibody. Particularly preferred ligand and antibodies are those thatbind to surface markers of immune cells. Chimeric molecules utilizingsuch antibodies as effector molecules act as bifunctional linkersestablishing an association between the immune cells bearing bindingpartner for the ligand or antibody and the tumor cells expressing thec-erbB-2. Suitable antibodies and growth factors are known to those ofskill in the art and include, but are not limited to, IL-2, IL-4, IL-6,IL-7, tumor necrosis factor (TNF), anti-Tac, TGF∀, and the like.

D) Other Therapeutic Moieties.

Other suitable effector molecules include pharmacological agents orencapsulation systems containing various pharmacological agents. Thus,the F5 or C1 antibody may be attached directly to a drug that is to bedelivered directly to the tumor. Such drugs are well known to those ofskill in the art and include, but are not limited to, doxirubicin,vinblastine, genistein, antisense molecules, ribozymes and the like.

Alternatively, the effector molecule may comprise an encapsulationsystem, such as a liposome, polymer capsule, viral capsid, virus, ormicelle that contains a therapeutic composition such as a drug, anucleic acid (e.g. an antisense nucleic acid including, but not limitedto a protein nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735 andConnor et al. (1985) Pharm. Ther., 28: 341-365.

In a particularly preferred embodiment, the liposome is a “stealth”(sterically stabilized) liposome bearing an external hydrophilic polymer(e.g. polyethylene glycol (PEG). The antibody can be coupled to theliposome through the hydrophilic polymer as described in copendingapplication U.S. Ser. No. 08/665,202.

E) Attachment of the F5 or C1 Antibody to the Effector Molecule.

One of skill will appreciate that the F5 or C1 antibody and the effectormolecule may be joined together in any order. Thus the effector moleculemay be joined to either the amino or carboxy termini of the F5 or C1antibody. The F5 or C1 antibody may also be joined to an internal regionof the effector molecule, or conversely, the effector molecule may bejoined to an internal location of the F5 or C1 antibody as long as theattachment does not interfere with the respective activities of themolecules.

The F5 or C1 antibody and the effector molecule may be attached by anyof a number of means well known to those of skill in the art. Typicallythe effector molecule is conjugated, either directly or through a linker(spacer), to the F5 or C1 antibody. However, where the effector moleculeis a polypeptide it is preferable to recombinantly express the chimericmolecule as a single-chain fusion protein.

i) Conjugation of the Effector Molecule to the F5 or C1 Antibody.

In one embodiment, the targeting molecule F5 or C1 antibody ischemically conjugated to the effector molecule (e.g. a cytotoxin, alabel, a ligand, or a drug or liposome). Means of chemically conjugatingmolecules are well known to those of skill (see, for example, Chapter 4in Monoclonal Antibodies: Principles and Applications, Birch and Lennox,eds. John Wiley & Sons, Inc. N.Y. (1995) which describes conjugation ofantibodies to anticancer drugs, labels including radio labels, enzymes,and the like).

The procedure for attaching an agent to an antibody or other polypeptidetargeting molecule will vary according to the chemical structure of theagent. Polypeptides typically contain variety of functional groups;e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, which areavailable for reaction with a suitable functional group on an effectormolecule to bind the effector thereto.

Alternatively, the targeting molecule and/or effector molecule may bederivatized to expose or attach additional reactive functional groups.The derivatization may involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, is a molecule that is used to join thetargeting molecule to the effector molecule. The linker is capable offorming covalent bonds to both the targeting molecule and to theeffector molecule. Suitable linkers are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Wherethe targeting molecule and the effector molecule are polypeptides, thelinkers may be joined to the constituent amino acids through their sidegroups (e.g., through a disulfide linkage to cysteine). However, in apreferred embodiment, the linkers will be joined to the alpha carbonamino and carboxyl groups of the terminal amino acids.

A bifunctional linker having one functional group reactive with a groupon a particular agent, and another group reactive with an antibody, maybe used to form the desired immunoconjugate. Alternatively,derivatization may involve chemical treatment of the targeting molecule,e.g., glycol cleavage of a sugar moiety attached to the protein antibodywith periodate to generate free aldehyde groups. The free aldehydegroups on the antibody may be reacted with free amine or hydrazinegroups on an agent to bind the agent thereto. (See U.S. Pat. No.4,671,958). Procedures for generation of free sulfhydryl groups onpolypeptide, such as antibodies or antibody fragments, are also known(See U.S. Pat. No. 4,659,839).

Many procedure and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known. See, for example, European Patent ApplicationNo. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. Cancer Res.47: 4071-4075 (1987) which are incorporated herein by reference. Inparticular, production of various immunotoxins is well-known within theart and can be found, for example in “Monoclonal Antibody-ToxinConjugates: Aiming the Magic Bullet,” Thorpe et al., MonoclonalAntibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982),Waldmann, Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and4,894,443.

In some circumstances, it is desirable to free the effector moleculefrom the targeting molecule when the chimeric molecule has reached itstarget site. Therefore, chimeric conjugates comprising linkages whichare cleavable in the vicinity of the target site may be used when theeffector is to be released at the target site. Cleaving of the linkageto release the agent from the antibody may be prompted by enzymaticactivity or conditions to which the immunoconjugate is subjected eitherinside the target cell or in the vicinity of the target site. When thetarget site is a tumor, a linker which is cleavable under conditionspresent at the tumor site (e.g. when exposed to tumor-associated enzymesor acidic pH) may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers that are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient's complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

ii) Production of Fusion Proteins.

Where the F5 or C1 antibody and/or the effector molecules are relativelyshort (i.e., less than about 50 amino acids) they may be synthesizedusing standard chemical peptide synthesis techniques. Where bothmolecules are relatively short the chimeric molecule may be synthesizedas a single contiguous polypeptide. Alternatively the F5 or C1antibodies and the effector molecule may be synthesized separately andthen fused by condensation of the amino terminus of one molecule withthe carboxyl terminus of the other molecule thereby forming a peptidebond. Alternatively, the targeting and effector molecules may each becondensed with one end of a peptide spacer molecule thereby forming acontiguous fusion protein.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

In a preferred embodiment, the chimeric fusion proteins of the presentinvention are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins (e.g. F5 or C1 Ab-PE) of this inventionmay be prepared by any suitable method, including, for example, cloningand restriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et al. (1979) Meth.Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979)Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method ofBeaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solidsupport method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

In a preferred embodiment, DNA encoding fusion proteins of the presentinvention may be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, the gene for the F5or C1 antibody may be amplified from a nucleic acid template (clone)using a sense primer containing a first restriction site and anantisense primer containing a second restriction site. This produces anucleic acid encoding the mature F5 or C1 antibody sequence and havingterminal restriction sites. A cytotoxin (or other polypeptide effector)may be cut out of a plasmid encoding that effector using restrictionenzymes to produce cut ends suitable for annealing to the F5 or C1antibody. Ligation of the sequences and introduction of the constructinto a vector produces a vector encoding the F5- or C1-effector moleculefusion protein. Such PCR cloning methods are well known to those ofskill in the art (see, for example, Debinski et al. (1994) Int. J.Cancer, 58: 744-748, for an example of the preparation of a PE fusionprotein).

While the two molecules may be directly joined together, one of skillwill appreciate that the molecules may be separated by a peptide spacerconsisting of one or more amino acids. Generally the spacer will have nospecific biological activity other than to join the proteins or topreserve some minimum distance or other spatial relationship betweenthem. However, the constituent amino acids of the spacer may be selectedto influence some property of the molecule such as the folding, netcharge, or hydrophobicity. One of skill will appreciate that PCR primersmay be selected to introduce an amino acid linker or spacer between theF5 or C1 antibody and the effector molecule if desired.

The nucleic acid sequences encoding the fusion proteins may be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost. For E. coli this includes a promoter such as the T7, trp, orlambda promoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, the control sequences willinclude a promoter and preferably an enhancer derived fromimmunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylationsequence, and may include splice donor and acceptor sequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). In a preferred embodiment, the fusion proteins arepurified using affinity purification methods as described in Examples 1and 2. Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the F5 or C1 antibody-effectorfusion protein may possess a conformation substantially different thanthe native conformations of the constituent polypeptides. In this case,it may be necessary to denature and reduce the polypeptide and then tocause the polypeptide to re-fold into the preferred conformation.Methods of reducing and denaturing proteins and inducing re-folding arewell known to those of skill in the art. (See, Debinski et al. J. Biol.Chem. (1993) 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4: 581-585; and Buchner, et al. (1992) Anal. Biochem., 205:263-270. Debinski et al., for example, describe the denaturation andreduction of inclusion body proteins in guanidine-DTE. The protein isthen refolded in a redox buffer containing oxidized glutathione andL-arginine.

One of skill would recognize that modifications can be made to the F5 orC1 antibody-effector fusion proteins without diminishing theirbiological activity. Some modifications may be made to facilitate thecloning, expression, or incorporation of the targeting molecule into afusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids placedon either terminus to create conveniently located restriction sites ortermination codons.

VI. Diagnostic Assays.

As explained above, the F5 or C1 antibodies may be used for the in vivoor in vitro detection and/or quantitation of c-erbB-2 and thus, in thediagnosis and/or localization of cancers characterized by the expressionof c-erbB-2.

A) In Vivo Detection of c-erB-2.

The F5 and C1 antibodies and/or chimeric molecules of the presentinvention may be used for in vivo detection and localization of cells(e.g. c-erbB-2 positive carcinoma) bearing c-erbB-2. Such detectioninvolves administering to an organism a chimeric molecule comprising aF5 or C6 antibody joined to a label detectable in vivo. Such labels arewell known to those of skill in the art and include, but are not limitedto, electron dense labels such as gold or barium which may be detectedby X-ray or CAT scan, various radioactive labels that may be detectedusing scintillography, and various magnetic and paramagnetic materialsthat may be detected using positron emission tomography (PET) andmagnetic resonance imaging (MRI). The F5 or C1 antibody associates thelabel with the c-erbB-2 bearing cell which is then detected andlocalized using the appropriate detection method.

B) In Vitro Detection of c-erB-2.

The F5 and C1 antibodies of this invention are also useful for thedetection of c-erbB-2 in vitro e.g., in biological samples obtained froman organism. The detection and/or quantification of c-erbB-2 in such asample is indicative the presence or absence or quantity of cells (e.g.,tumor cells) overexpressing c-erbB-2.

The c-erbB-2 antigen may be quantified in a biological sample derivedfrom a patient such as a cell, or a tissue sample derived from apatient. As used herein, a biological sample is a sample of biologicaltissue or fluid that contains a c-erbB-2 antigen concentration that maybe correlated with and indicative of cells overexpressing c-erbB-2.Preferred biological samples include blood, urine, and tissue biopsies.

In a particularly preferred embodiment, erB-2 is quantified in breasttissue cells derived from normal or malignant breast tissue samples.Although the sample is typically taken from a human patient, the assayscan be used to detect erB-2 in cells from mammals in general, such asdogs, cats, sheep, cattle and pigs, and most particularly primates suchas humans, chimpanzees, gorillas, macaques, and baboons, and rodentssuch as mice, rats, and guinea pigs.

Tissue or fluid samples are isolated from a patient according tostandard methods well known to those of skill in the art, most typicallyby biopsy or venipuncture. The sample is optionally pretreated asnecessary by dilution in an appropriate buffer solution or concentrated,if desired. Any of a number of standard aqueous buffer solutions,employing one of a variety of buffers, such as phosphate, Tris, or thelike, at physiological pH can be used.

C) Assay Formats (Detection or Quantification of c-erbB-2).

1) Immunological Binding Assays

The c-erB-2 peptide (analyte) or an anti-c-erb-2 antibody is preferablydetected in an immunoassay utilizing a F5 or C1 antibody as a captureagent that specifically binds to a c-erbB-2 peptide.

As used herein, an immunoassay is an assay that utilizes an antibody(e.g. a F5 or C1 antibody) to specifically bind an analyte (e.g.,c-erb-2). The immunoassay is characterized by the use of specificbinding to a F5 or C1 antibody as opposed to other physical or chemicalproperties to isolate, target, and quantify the c-erB-2 analyte.

The c-erbB-2 marker may be detected and quantified using any of a numberof well recognized immunological binding assays. (See for example, U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168) For a reviewof the general immunoassays, see also Methods in Cell Biology Volume 37:Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York(1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds.(1991)).

The immunoassays of the present invention are performed in any ofseveral configurations, e.g., those reviewed in Maggio (ed.) (1980)Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985) “Practiceand Theory of Enzyme Immunoassays,” Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers B. V.,Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987) Immunoassay. APractical Guide Academic Press, Orlando, Fla.; Price and Newman (eds.)(1991) Principles and Practice of Immunoassays Stockton Press, NY; andNgo (ed.) (1988) Non isotopic Immunoassays Plenum Press, NY.

Immunoassays often utilize a labeling agent to specifically bind to andlabel the binding complex formed by the capture agent and the analyte(i.e., a F5 or C1 antibody-erB-2 complex). The labeling agent may itselfbe one of the moieties comprising the antibody/analyte complex. Thus,the labeling agent may be a labeled c-erB-2 peptide or a labeled F5 orC1 antibody. Alternatively, the labeling agent is optionally a thirdmoiety, such as another antibody, that specifically binds to the F5 orC1 antibody, the c-erB-2 peptide, the anti-c-erB-2 antibody/c-erB-2peptide complex, or to a modified capture group (e.g., biotin) which iscovalently linked to c-erB-2 or the F5 or C1 antibody.

In one embodiment, the labeling agent is an antibody that specificallybinds to the F5 or C1 antibody. Such agents are well known to those ofskill in the art, and most typically comprise labeled antibodies thatspecifically bind antibodies of the particular animal species from whichthe F5 or C1 antibody is derived (e.g., an anti-species antibody). Thus,for example, where the capture agent is a human derived F5 or C1antibody, the label agent may be a mouse anti-human IgG, i.e., anantibody specific to the constant region of the human antibody.

Other proteins capable of specifically binding immunoglobulin constantregions, such as streptococcal protein A or protein G are also used asthe labeling agent. These proteins are normal constituents of the cellwalls of streptococcal bacteria. They exhibit a strong non immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies. See, generally Kronval, et al., (1973) J. Immunol.,111:1401-1406, and Akerstrom, et al., (1985) J. Immunol., 135:2589-2542.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays are carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 5° C. to 45° C.

a) Non Competitive Assay Formats.

Immunoassays for detecting c-erb-2 are typically either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte (in this case, c-erb-2) is directly measured.In one preferred “sandwich” assay, for example, the capture agent (e.g.,F5 or C1 antibody) is bound directly or indirectly to a solid substratewhere it is immobilized. These immobilized F5 or C1 antibodies capturec-erb-2 present in a test sample (e.g., a biological sample derived frombreast tumor tissue). The c-erb-2 thus immobilized is then bound by alabeling agent, such as a second c-erb-2 antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. Free labeled antibodyis washed away and the remaining bound labeled antibody is detected(e.g., using a gamma detector where the label is radioactive). One ofskill will appreciate that the analyte and capture agent is optionallyreversed in the above assay, e.g., when the presence, quantity oravidity of a F5 or C1 antibody in a sample is to be measured by itsbinding to an immobilized c-erb-2 peptide.

b) Competitive Assay Formats.

In competitive assays, the amount of analyte (e.g., c-erB-2) present inthe sample is measured indirectly by measuring the amount of an added(exogenous) analyte displaced (or competed away) from a capture agent(e.g., F5 or C1 antibody) by the analyte present in the sample. In onecompetitive assay, a known amount of c-erb-2 is added to a test samplewith an unquantified amount of c-erB-2, and the sample is contacted witha capture agent, e.g., a F5 or C1 antibody that specifically bindsc-erb-2. The amount of added c-erB-2 which binds to the F5 or C1antibody is inversely proportional to the concentration of c-erB-2present in the test sample.

The F5 or C1 antibody can be immobilized on a solid substrate. Theamount of erB-2 bound to the F5 or C1 antibody is determined either bymeasuring the amount of erB-2 present in an erB-2-C6 antibody complex,or alternatively by measuring the amount of remaining uncomplexed erB-2.Similarly, in certain embodiments where the amount of erB-2 in a sampleis known, and the amount or avidity of a F5 or C1 antibody in a sampleis to be determined, erB-2 becomes the capture agent (e.g., is fixed toa solid substrate) and the F5 or C1 antibody becomes the analyte.

c) Reduction of Non Specific Binding.

One of skill will appreciate that it is often desirable to reduce nonspecific binding in immunoassays and during analyte purification. Wherethe assay involves c-erB-2, F5 or C1 antibody, or other capture agentimmobilized on a solid substrate, it is desirable to minimize the amountof non specific binding to the substrate. Means of reducing such nonspecific binding are well known to those of skill in the art. Typically,this involves coating the substrate with a proteinaceous composition. Inparticular, protein compositions such as bovine serum albumin (BSA),nonfat powdered milk, and gelatin are widely used.

d) Substrates.

As mentioned above, depending upon the assay, various components,including the erB-2, F5 or C1 or antibodies to erB-2 or F5 or C1, areoptionally bound to a solid surface. Many methods for immobilizingbiomolecules to a variety of solid surfaces are known in the art. Forinstance, the solid surface may be a membrane (e.g., nitrocellulose), amicrotiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube(glass or plastic), a dipstick (e.g. glass, PVC, polypropylene,polystyrene, latex, and the like), a microcentrifuge tube, or a glass,silica, plastic, metallic or polymer bead. The desired component may becovalently bound, or noncovalently attached through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

In preparing the surface, a plurality of different materials may beemployed, e.g., as laminates, to obtain various properties. For example,protein coatings, such as gelatin can be used to avoid non specificbinding, simplify covalent conjugation, enhance signal detection or thelike.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas, J. Biol. Chem. 245 3059(1970).

In addition to covalent bonding, various methods for noncovalentlybinding an assay component can be used. Noncovalent binding is typicallynonspecific absorption of a compound to the surface. Typically, thesurface is blocked with a second compound to prevent nonspecific bindingof labeled assay components. Alternatively, the surface is designed suchthat it nonspecifically binds one component but does not significantlybind another. For example, a surface bearing a lectin such asconcanavalin A will bind a carbohydrate containing compound but not alabeled protein that lacks glycosylation. Various solid surfaces for usein noncovalent attachment of assay components are reviewed in U.S. Pat.Nos. 4,447,576 and 4,254,082.

2) Other Assay Formats

C-erB-2 polypeptides or F5 or C1 antibodies and can also be detected andquantified by any of a number of other means well known to those ofskill in the art. These include analytic biochemical methods such asspectrophotometry, radiography, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like.

Western blot analysis and related methods can also be used to detect andquantify the presence of erB-2 peptides and F5 or C1 antibodies in asample. The technique generally comprises separating sample products bygel electrophoresis on the basis of molecular weight, transferring theseparated products to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind eitherthe erB-2 peptide or the anti-erB-2 antibody. The antibodiesspecifically bind to the biological agent of interest on the solidsupport. These antibodies are directly labeled or alternatively aresubsequently detected using labeled antibodies (e.g., labeled sheepanti-human antibodies where the antibody to a marker gene is a humanantibody) which specifically bind to the antibody which binds eitheranti-erB-2 or erB-2 as appropriate.

Other assay formats include liposome immunoassays (LIAs), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,(1986) Amer. Clin. Prod. Rev. 5:34-41).

VII. Pharmaceutical Compositions.

The F5 and C1 antibodies and chimeric molecules of this invention areuseful for parenteral, topical, oral, or local administration, such asby aerosol or transdermally, for prophylactic and/or therapeutictreatment. The pharmaceutical compositions can be administered in avariety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, capsules and lozenges. Itis recognized that the fusion proteins and pharmaceutical compositionsof this invention, when administered orally, must be protected fromdigestion. This is typically accomplished either by complexing theprotein with a composition to render it resistant to acidic andenzymatic hydrolysis or by packaging the protein in an appropriatelyresistant carrier such as a liposome. Means of protecting proteins fromdigestion are well known in the art.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a solution of thechimeric molecule dissolved in a pharmaceutically acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions may besterilized by conventional, well-known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofchimeric molecule in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in such publications as Remington's PharmaceuticalScience, 15th ed., Mack Publishing Company, Easton, Pa. (1980).

The compositions containing the present fusion proteins or a cocktailthereof (i.e., with other proteins) can be administered for therapeutictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease, typically a c-erbB-2 positivecarcinoma, in an amount sufficient to cure or at least partially arrestthe disease and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Amounts effective forthis use will depend upon the severity of the disease and the generalstate of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

Among various uses of the cytotoxic fusion proteins of the presentinvention are included a variety of disease conditions caused byspecific human cells that may be eliminated by the toxic action of theprotein. One application is the treatment of cancer, such as by the useof a F5 or C1 antibody attached to a cytotoxin.

Another approach involves using a ligand that binds a cell surfacemarker (receptor) so the chimeric associates cells bearing the ligandsubstrate are associated with the c-erbB-2 overexpressing tumor cell.The ligand portion of the molecule is chosen according to the intendeduse. Proteins on the membranes of T cells that may serve as targets forthe ligand includes FcI, FcII and FcIII, CD2 (T11), CD3, CD4 and CD8.Proteins found predominantly on B cells that might serve as targetsinclude CD10 (CALLA antigen), CD19 and CD20. CD45 is a possible targetthat occurs broadly on lymphoid cells. These and other possible targetlymphocyte target molecules for the chimeric molecules bearing a ligandeffector are described in Leukocyte Typing III, A. J. McMichael, ed.,Oxford University Press (1987). Those skilled in the art will realizeligand effectors may be chosen that bind to receptors expressed on stillother types of cells as described above, for example, membraneglycoproteins or ligand or hormone receptors such as epidermal growthfactor receptor and the like.

VIII. Kits for Diagnosis or Treatment.

In another embodiment, this invention provides for kits for thetreatment of tumors or for the detection of cells overexpressingc-erbB-2. Kits will typically comprise a chimeric molecule of thepresent invention (e.g. F5 and/or C1 antibody-label, F5 and/or C1antibody-cytotoxin, F5 and/or C1 antibody-ligand, etc.). In addition thekits may optionally include instructional materials containingdirections (i.e., protocols) disclosing means of use of the chimericmolecule(s) (e.g. as a cytotoxin, for detection of tumor cells, toaugment an immune response, etc.). While the instructional materialstypically comprise written or printed materials they are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this invention. Such mediainclude, but are not limited to electronic storage media (e.g., magneticdiscs, tapes, cartridges, chips), optical media (e.g., CD ROM), and thelike. Such media may include addresses to internet sites that providesuch instructional materials.

The kits may also include additional components to facilitate theparticular application for which the kit is designed. Thus, for example,where a kit contains a chimeric molecule in which the effector moleculeis a detectable label, the kit may additionally contain means ofdetecting the label (e.g. enzyme substrates for enzymatic labels, filtersets to detect fluorescent labels, appropriate secondary labels such asa sheep anti-human antibodies, or the like). The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Creation of a Non-Immune Human Fab Phage Antibody LibraryContaining 10⁹-10¹¹ Members

Manipulation of previous 10⁷ member phage display libraries revealed twomajor limitations: 1) expression levels of Fabs was too low to produceadequate material for characterization, and 2) the library wasrelatively unstable. These limitations are a result of creating thelibrary in a phage vector, and the use of the cre-lox recombinationsystem. We therefore decided that the best approach for this project wasto create a very large scfv library using a phagemid vector. The goalwas to produce a library at least 100 times larger than our previous3.0×10⁷ member scFv library. The approach taken was to clone the V_(H)and V_(L) library on separate replicons, combine them into an scFv generepertoire by splicing by overlap extension, and clone the scFv generepertoire into the phage display vector pHEN1. Human peripheral bloodlymphocyte and spleen RNA was primed with IgM heavy chain constantregion and, kappa and lambda light chain constant region primers andfirst strand cDNA synthesized. 1st strand cDNA was used as a templatefor PCR amplification of VH Vκk and Vλ gene repertoires.

The V_(H) gene repertoires were cloned into the vector pUC119Sfi-Not asNco1-NotI fragments, to create a library of 8.0×108 members. The librarywas diverse by PCR fingerprinting. Single chain linker DNA was splicedonto the V_(L) gene repertoires using PCR- and the repertoire cloned asan XhoI-NotI fragment into the vector pHENIXscFv to create a library of7.2×10⁶ members. The V_(H) and V_(L) gene repertoires were amplifiedfrom their respective vectors and spliced together using PCR to createan scFv gene repertoire. The scFv gene repertoire was cloned as anNcoI-NotI fragment into the vector to create an scFv phage antibodylibrary of 7.0×10⁹ members. The library was diverse as determined byBstN1 fingerprinting.

To verify the quality of the library, phage were prepared and selectedon 14 different protein antigens. The results are shown in Table 1. scFvantibodies were obtained against all antigens used for selection, withbetween 3 and

TABLE 1 Results of phage antibody library selections. For each antigen(column 1), the number and the percentage of positive clones selected(column 2) and the number of different antibodies isolated (column 3) isindicated Number of different Percentage (number) of antibodies Proteinantigen used for selection ELISA positive clones isolated FGF ReceptorECD 69 (18/26) 15 BMP Receptor Type I ECD 50 (12/24) 12 Activin ReceptorType I ECD 66 (16/24) 7 Activin Receptor Type II ECD 66 (16/24) 4 Erb-B2ECD 91 (31/34) 14 VEGF 50 (48/96) 6 BoNT/A 28 (26/92) 14 BoNT-AC-fragment 95 (87/92) 10 BoNT/B 10 (9/92) 5 BoNT/C 12 (11/92) 5 BoNT/E 9 (8/92) 3 Bungarotoxin 67 (64/96) 15 Cytochrome b5 55 (53/96) 5Chlamydia trachomatis EB 66 (63/96) 715 unique scFv isolated per antigen (average 8.7) (Table 1). Thiscompares favorably to results obtained from smaller scFv libraries (1 toa few binders obtained against only 70% of antigens used for selection).Affinities of 4 anti-ErbB-2 scFv and 4 anti-Botulinum scFv were measuredusing surface plasmon resonance in a BIAcore and found to range from4.0×10⁻⁹M to 2.2×10⁻¹⁰ M for the anti-ErbB2 scFv and 2.6×10⁻⁸ M to7.15×10⁻⁸ M for the anti-Botulinum scFv (Table 2). scFv were highlyspecific for the antigen used for selection (FIG. 2). The library couldalso be successfully selected on complex mixtures of antigen.

TABLE 2 Affinities and binding kinetics of anti-BoNT A C-fragment andanti-Erb-B2 scFv. Association (k_(on)) and dissociation (k_(off)) rateconstants for purified scFvs were measured using surface plasmonresonance (BIAcore) and K_(d) calculated as (k_(off)/k_(on)).Specificity and clone K_(d) (×10⁻⁹ M) k_(on) (×10⁵ M⁻¹s⁻¹) k_(off)(×10⁻³ s⁻¹) ErbB-2 B7A 0.22 4.42 0.1 ErbB-2 G11D 0.48 2.19 0.11 ErbB-2A11A 0.49 3.69 0.18 ErbB-2 F5A 4.03 1.62 0.65 BoNT-A 2A9 26.1 0.25 0.66BoNT-A 2H6 38.6 2.2 8.5 BoNT-A 3F6 66.0 4.7 30.9 BoNT-A 2B6 71.5 1.1 7.8For example, selection on Chlamydia trachomatis elementary bodies (thecausative organism of Chlamydial disease) yielded seven thatspecifically recognized chlamydia (Table 1 and FIG. 3). The scFv couldbe successfully used in a number of immunologic assays including ELISA,immunofluorescence, Western blotting, epitope mapping andimmunoprecipitation. The number of binding antibodies for each antigen,and the affinities of the binding scFv are comparable to resultsobtained from the best phage antibody libraries (Table 3). Thus thelibrary was established as a source of panels of human antibodiesagainst any antigen with affinities at least equivalent to the secondarymurine response.

TABLE 3 Comparison of protein binding antibodies selected fromnon-immune phage- display antibody libraries. Average Range of Numbernumber of Number affinities for of protein antibodies of protein Librarysize and antigens per protein affinities antigens Library type* studiedantigen measured K_(d) (×10⁻⁹ M) Marks et al (1991) J. 3.0 × 10⁷ (scFv,N) 2 2.5 1 100-2000 Mol. Biol. 222: 581-597 Nissim et al (1994) 1.0 ×10⁸ (scFV, SS) 15 2.6 ND ND EMBO J. 13: 692-698 DeKruif et al (1995) J.3.6 × 10⁸ (scFv, SS) 12 1.9 3 100-2500 Mol. Biol. 248: 97-105 Griffithset al (1994) 6.5 × 10¹⁰ (Fab, SS) 30 4.8 3 7-58 EMBO J. 13: 3245-3260Vaughan et al (1996) 1.4 × 10¹⁰ (scFv, N) 3 7.0 3 4.2-8.0  NatureBiotechnology. 14: 309-314 Present Examples 6.7 × 10⁹ (scFv, N) 14 8.7 80.22-71.5  *For library type, N = V-gene repertoires obtained fromV-genes rearranged in vivo; SS = semi-synthetic V-genes constructed fromcloned V-gene segments and synthetic oligonucleotides encoding V_(H)CDR3. ND = not determined.

These experiments demonstrate the creation of a high complexity humanscFv phage antibody library from which a panel of high affinity humanscFv can be generated against any purified antigen. Such a library isideal for probing the surface of cells to identify novel cell surfacemarkers.

Example 2 Uptake of scFV into Cells by Receptor Mediated Endocytosis andSubsequent Recovery

The 7.0×10⁹ member scFv phage antibody library described above wasselected on the malignant breast tumor cell lines MB231 and ZR-75-1,both with and without negative selections on the normal breast cell lineHBL100. Similar results were obtained as described in section above.scFv were isolated that could not distinguish malignant fromnon-malignant cell lines.

To increase the specificity of selections, it was hypothesized thatphage binding cell surface receptors could be taken up into cells byreceptor mediated endocytosis and could then be recovered from cells bylysing the cells. This assumed: 1) that phage could be internalized byreceptor mediated endocytosis and 2) that phage could be recovered inthe infectious state from within cells prior to lysosomal degradation.The ability to select for internalized phage antibodies would have twomajor benefits: 1) the identification of antibodies that bind toreceptors capable of internalization and 2) an added level ofspecificity in the selection process. Identification of antibodies whichare internalized would be highly useful for many targeted therapeuticapproaches where internalization is essential (e.g. immunotoxins,targeted liposomes, targeted gene therapy vectors and others).

A) Receptor Mediated Internalization of F5 or C1 Phage

To determine proof of principle, we utilized C6.5 phage and C6.5 diabodyphage (see, copending application U.S. Ser. No. 08/665,202). We havepreviously shown that C6.5 scFv is internalized, but at a slow rate, andthat the C6.5 diabody is somewhat better internalized (probably becauseit causes receptor dimerization). C6.5 phage, C6.5 diabody phage or anirrelevant anti-Botulinum phage were incubated with SKBR3 cells (ErbB2expressing breast tumor cell line) at either 37° C. or 4° C. andnon-internalized phage removed by sequential washing with PBS and low pHglycine buffer. The cells were then permeabilized and biotinylatedanti-M13-antibody added followed by streptavidin Texas Red. Cells werethen examined by using a confocal microscope. Both C6.5 phage and C6.5diabody phage were observed within the cytoplasm). Approximately 1% ofcells had internalized C6.5 phage and 20% of the cells had internalizedC6.5 diabody phage. There was no internalization of the anti-Botulinumphage.

To determine if infectious phage could be specifically taken up andrecovered from within cells, C6.5 phage or C6.5 diabody phage wereincubated with SKBR3 cells at 37° C. Non bound phage were removed bywashing with PBS and phage bound to the cell surface were eluted bywashing twice with low pH glycine. The cells were then lysed and eachfraction (the first and second glycine washes and the cytoplasmicfraction) used to infect E. coli TG1. Twenty times (C6.5) or 30 times(C6.5 diabody) more phage were bound to the cell surface than theanti-Botulinum phage (glycine 1 wash) (Table 4). After the secondglycine wash, the titre of infectious phage from the cell surfacedecreased, indicating that washing was effective at removing surfacebound phage (Table 4). After cell lysis, the titer increased more than10 fold (C6.5 phage) or 50 fold (C6.5 diabody phage) from the secondglycine wash. We believe this titre represents phage recovered frominside the cell. Recovery of phage from inside the cell was 100 timeshigher for ErbB2 binding C6.5 than for anti-Botulinum phage and 200 foldhigher for C6.5 diabody phage (Table 4).

TABLE 4 Titer of cell surface bound phage and internalized phage. 5.0 ×10¹¹ phage (anti-Botulinum or anti-ErbB2) were incubated withapproximately 1.0 × 10⁵ ErbB2 expressing SKBR3 cells at 37° C. Cellswere washed 10 times with PBS and surface bound phage eluted with twolow pH glycine washes. The cells were then washed once with PBS and thecells lysed to release internalized phage. The phage titer was thendetermined for each of the glycine washes and for the lysed cellfraction by infection of E. coli TG1. 2nd glycine Lysed cell Phagespecificity 1st glycine wash wash fraction anti-Botulinum 6.0 × 10⁵ 1.0× 10⁵ 6.0 × 10⁵ Anti-ErbB2 (C6.5 scFv) 1.2 × 10⁷ 5.2 × 10⁶ 6.8 × 10⁷Anti-ErbB2 (C6.5 diabody) 1.8 × 10⁷ 2.8 × 10⁶ 1.7 × 10⁷

Taken together, the results indicate that: 1) phage binding cell surfacereceptors can be taken up by cells and the infectious phage recoveredfrom the cytoplasm. The amount of uptake is significantly greater thanuptake of non-binding phage, and the 100 to 200 fold difference is wellwithin the range that would allow enrichment from a library. What isunknown from the results is whether the phage antibodies are mediatingreceptor mediated internalization or whether they are merely taken upafter binding by membrane turnover.

B) Selection and Characterization of Internalizing Antibodies from aPhage Antibody Library

The results described above encouraged us to attempt selection of thephage antibody library described above to identify new phage antibodiesthat were internalized. Phage antibodies were rescued from the libraryand selected on SKBR3 cells. For selection, phage were incubated withcells at 37° C., non-binding phage removed by washing cells with PBS andphage bound to cell surface antigens removed by sequential washes withlow pH glycine. Cells were then lysed to release internalized phage andthe lysate used to infect E. coli TG1 to prepare phage for the nextround of selection. Three rounds of selection were performed. Onehundred clones from each round of selection were analyzed for binding toSKBR3 cells and to ErbB2 extracellular domain by ELISA. We hypothesizedthat we were likely to obtain binders to ErbB2 since SKBR3 cells areknown to express high levels and ErbB2 is a receptor which is known tobe internalized. After each round of selection, the titer of phagerecovered from the cytoplasm increased (Table 5). After the third round,45% of the clones were positive SKBR3 cell binding and 17% bound ErbB2(Table 5).

TABLE 5 Results of selection of a phage antibody library forinternalization. For each round of selection, the titer of phage inlysed cells, number of cells lysed and number of phage per cell isindicated. After the third round, individual clones were analyzed forbinding to SKBR3 cells by ELISA and to ErbB2 ECD by ELISA. Round of # ofphage in # of cells # of % SKBR3 % ErbB2 selection cell lysate lysedphage/cell binders binders 1 3.5 × 10⁴ 2.8 × 10⁶ 0.013 ND ND 2 1.2 × 10⁵2.8 × 10⁶ 0.038 ND ND 3 7.5 × 10⁶ 2.8 × 10⁶ 3.75 45% 17%

To estimate the number of unique binders, the scFv gene from ELISApositive clones was PCR amplified and fingerprinted by digestion withBstN1. Two unique restriction patterns were identified. The scFv geneswere sequenced and 2 unique ErbB2 binding scFv identified. Similaranalysis of SKBR3 ELISA positive clones that did not bind ErbB2identified an additional 11 unique scFv.

To verify that phage antibodies were specific for SKBR3 cells, phagewere prepared from each unique clone and analyzed for binding to SKBR3cells (high ErbB2 expression) as well as 2 other epithelial tumor celllines (SK-OV-3, moderate ErbB2 expression and MCF7, low ErbB2expression) and a normal breast cell line (HS578B). Each unique clonespecifically stained tumor cell lines but not the normal breast cellline.

SKBR3 and MCF7 cells were incubated with phage antibodies C6.5 (positivecontrol), 3TF5 and 3 GH7. The latter two clones were isolated from thelibrary, with 3TF5 binding ErbB2 and the antigen bound by 3 GH7 unknown.All 3 phage antibodies intensely stain SKBR3 cells (the selecting cellline and high ErbB2 expresser. C6.5 phage weakly stain MCF7 cells (lowErbB2 expressor). The anti-ErbB2 clone 3TF5 from the library stains MCF7cells much more intensely then C6.5, as does 3 GH7.

SKBR3, SK-OV-3, MCF7 and HST578 cells were studied using native purifiedscFv 3TF5 and 3 GH7. For these studies, the scFv genes were subclonedinto a vector which fuses a hexahistidine tag to the scFv C-terminus.scFv was then expressed, harvested from the bacterial periplasm andpurified by immobilized metal affinity chromatography. The two scFvintensely stain SKBR3 cells, and do not stain the normal breast cellline HST578. There is minimal staining of the low ErbB2 expressing cellline MCF7 and intermediate staining of SK-OV-3 cells (moderate ErbB2expresser). In general, the intensity of staining is less than seen withphage. This is to be expected since the secondary antibody for phagestaining recognizes the major coat protein (2500 copies/phage) resultingin tremendous signal amplification.

The anti-ErbB2 phage antibody 3TF5 was studied further to determine ifit was indeed internalized. This antibody was selected for initial studysince its internalization could be compared to ErbB2 binding C6.5.5.0×10¹¹ 3TF5 or C6.5 phage were incubated with SKBR3 cells at 37° C. orat 4° C. After washing with PBS, 3TF5 phage stained cells more intenselythan C6.5 phage. After washing with low pH glycine, confocal microscopyrevealed that 3TF5 phage were internalized by greater than 95% of cells,while C6.5 was internalized by only a few percent of cells. Incubationof either antibody at 4° C. led to no internalization.

The native purified 3TF5 scFv was similarly analyzed and was alsoefficiently internalized by SKBR3 cells. It should be noted that thenative 3TF5 scFv existed only as a monomer with no appreciabledimerization or aggregation as determined by gel filtration.

These experiments demonstrate that phage antibodies can be internalizedby cells and recovered from the cytoplasm. Phage that bind aninternalizing cell surface receptor can be enriched more than 100 foldover non-binding phage. This level of enrichment is greater than thatachieved by selecting on the cell surface. We have applied this approachto library selection and isolated phage antibodies that bind and areinternalized by SKBR-3 cells. Several of these antibodies bind to ErbB2,but are more efficiently internalized than antibodies such as C6.5 thatwere generated by selecting on pure antigen. Many other antibodies havebeen isolated that bind specifically to SKBR-3 and other breast tumorcell lines and are efficiently internalized. These antibodies shouldprove useful for tumor targeting and for identifying potentially novelinternalizing tumor cell receptors.

Example 3 Increasing the Affinity of Antibody Fragments with the DesiredBinding Characteristics by Creating Mutant Phase Antibody Libraries andSelecting on the Appropriate Breast Tumor Cell Line

Phage display has the potential to produce antibodies with affinitiesthat cannot be produced using conventional hybridoma technology. Ultrahigh affinity human antibody fragments could result in excellent tumorpenetration, prolonged tumor retention, and rapid clearance from thecirculation, leading to high specificity. We therefore undertook aseries of experiments to develop methodologies to generate ultra highaffinity human antibody fragments. Experiments were performed to answerthe following questions: 1) What is the most effective way to select andscreen for rare higher affinity phage antibodies amidst a background oflower affinity binders; 2 What is the most effective means to removebound phage from antigen, to ensure selection of the highest affinityphage antibodies; 3) What is the most efficient techniques for makingmutant phage antibody libraries (random mutagenesis or site directedmutagenesis; 4) What region of the antibody molecule should be selectedfor mutagenesis to most efficiently increase antibody fragment affinity.

To answer these questions, we studied the human scFv C6.5, which bindsthe extracellular domain (ECD) of the tumor antigen ErbB-2 (32) with aK_(d) of 1.6×10⁻⁸ M and k_(off) of 6.3×10⁻³ s⁻¹ (Schier et al. (1995)Immunotechnology, 1: 63-71). Isolation and characterization of C6.5 isdescribed briefly below and in detail in copending application U.S. Ser.No. 08/665,202).

Despite excellent tumor:normal tissue ratios in vivo, quantitativedelivery of C6.5 was not adequate to cure tumors in animals usingradioimmunotherapy (Schier et al. (1995) Immunotechnology, 1: 63-71). Toimprove the quantitative delivery of antibody to tumor, the affinity ofC6.5 was increased. First, techniques were developed that allowedselection of phage antibodies on the basis of affinity, rather thandifferential growth in E. coli or host strain toxicity (Schier et al.(1996) J. Mol. Biol. 255: 28-43; Schier et al. (1996) Gene 169: 147-155;Schier et al. (1996) Human antibodies and hybridomas 7: 97-105). Next,we determined which locations in the scFv gene to mutate to achieve thegreatest increments in affinity (Schier et al. (1996) J. Mol. Biol. 255:28-43; Schier et al. (1996) Gene; Schier et al. (1996) J. Mol. Biol.263: 551-567). Random mutagenesis did not yield as great an increment inaffinity as site directed mutagenesis of the complementarity determiningregions (CDRs) that contain the amino acids which contact antigen.Results from diversifying the CDRs indicated that: 1) the greatestincrement in affinity was achieved by mutating the CDRs located in thecenter of the binding pocket (V_(L) and V_(H) CDR3); 2) half of the CDRresidues have a structural role in the scFv and when mutated return aswild-type; and 3) these structural residues can be identified prior tolibrary construction by modeling on a homologous atomic crystalstructure. These observations led to development of a generic strategyfor increasing antibody affinity where mutations are randomly introducedsequentially into V_(H) and V_(L) CDR3, with conservation of residuespostulated to have a structural role by homology modeling (Schier et al.(1996) J. Mol. Biol. 263: 551-567). Using this approach, the affinity ofC6.5 was increased 1200 fold to a K_(d) of 1.3×10⁻¹¹ M (Id.).

Biodistribution studies revealed a close correlation between affinityand the percent injected dose of scFv/gram of tumor (% ID/g) at 24 hours(Adams et al. (1998) Cancer Res. 58: 485-490). The greatest degree oftumor retention was observed with ¹²⁵I-C6ML3-9 (1.42% ID/g,K_(d)=1.0×10⁻⁹ M). Significantly less tumor retention was achieved with¹²⁵I-C6.5 (0.80% ID/g, K_(d)=1.6×10⁻⁸) and C6G98A (0.19% ID/g,K_(d)=3.2×10⁻⁷ M). The tumor:normal organ ratios also reflected thedifferences in affinity, e.g. tumor:blood ratios of 17.2, 13.3, 3.5 and2.6, and tumor to liver ratios of 26.2, 19.8, 4.0 and 3.1 for C6ML3-9,C6.5 and C6G98A respectively at 24 hours. Studies of the higher affinityscFv are pending. The results demonstrate our ability to increaseantibody affinity to values not achievable from hybridoma technology andconfirm the importance of affinity in tumor targeting

Example 4 Preclinical Development of C6.5 Based Breast Cancer Therapies

Two approaches have been collaboratively pursued to develop C6.5 basedbreast cancer therapies. In one collaboration, C6.5 based molecules arebeing engineered for radioimmunotherapy. To increase quantitative tumordelivery and retention of antibody fragment, dimeric scFv ‘diabodies’were created by shortening the linker between the V_(H) and V_(L)domains from 15 to 5 amino acids. Consequently, pairing occurs betweencomplementary domains of two different chains, creating a stablenoncovalently bound dimer with two binding sites. In vitro, diabodiesproduced from the V-genes of C6.5 have a significantly higher apparentaffinity and longer retention on the surface of SK-OV-3 cells comparedto C6.5 scFv (T_(1/2)>5 hr vs. 5 min) (Adams et al. (1998) Brit. J.Cancer). Biodistribution studies of C6.5 diabody revealed 6.5% ID/gtumor at 24 hours compared to only 1% ID/g for C6.5 scFv. When diabodyretentions were examined over 72 hours and cumulative area under thecurve (AUC) values determined, the resulting tumor:organ AUC ratios weregreater than reported for other monovalent or divalent scFv molecules.The therapeutic potential of these molecules is being examined inradioimmunotherapy studies in nude mice. Since in vivo characterizationof c6.5 based molecules was not formally one of the technicalobjectives, we are continuing to use the affinity mutants of C6.5 andC6.5 based diabodies to study the relationship between antibodyaffinity, size and valency and specific tumor targeting as part of NIHR01 1 CA65559-01A1.

In a second collaboration, C6.5 based molecules are being used to targetdoxorubicin containing stealth liposomes to ErbB2 expressing breastcancers (Kirpotin et al. (1997) Biochemistry. 36: 66-75). To facilitatechemical coupling of the scFv to liposomes, the C6.5 gene was subclonedinto an E. coli expression vector resulting in addition of a freecysteine residue at the C-terminus of the scFv. Purified C6.5cys scFvwas conjugated to liposomes and in vitro uptake determined using SKBR3cells. Total uptake was 3.4 mmol phospholipid/10⁶ cells at 6 hour, with70% of the uptake internalized. The uptake is comparable to thatachieved using the 4D5 anti-HER2Fab′ from Genentech. There was no uptakeof unconjugated liposomes. The results indicate that C6.5 binds to aErbB2 epitope that results in internalization at a rate comparable tothe best internalizing antibody produced from hybridomas (4D5). In vivotherapy studies in scid mice indicated that C6.5 targeted liposomescaused a greater degree of tumor regression and a higher cure rate thanuntargeted liposomes or a combination of untargeted liposomes andsystemic 4D5 antibody.

CONCLUSIONS

The experiments described herein establish that A large (7.0×10⁹ member)phage antibody library has been created which can provide panels ofhuman antibodies to purified antigens with affinities comparable to theaffinities of antibodies produced by murine immunization. The phageantibodies binding cell surface receptors can be can be internalized bycells and recovered in an infectious state from within the cell.Methodologies were developed which permit enrichment of internalizingphage antibodies over non-internalizing antibodies more than 100 fold.These methodologies were then applied to select new scFv antibodies thatbind to internalizing receptors on SKBR-3 cells. Several of theseantibodies bind to ErbB2, but are internalized more efficiently thanC6.5 based scFv. Many more antibodies bind to unknown internalizingreceptors. All of these scFv bind specifically to SKBR-3 cells orrelated tumor cell lines. The results indicate that this selectionapproach is a powerful approach to generate antibodies that candistinguish one cell type (malignant) from another (non-malignant).Moreover, we have demonstrated that it is not only possible to selectfor binding, but to select for function (internalization). In the nearterm, we will further characterize the antibodies isolated with respectto specificity, and in the case of ErbB2 binding scfv, affinity. In thelonger term we will use these reagents to: 1) study the effect ofaffinity and valency on the rate of internalization; and 2) identify theantigens bound using immunoprecipitation. It is likely that the resultswill lead to the identification of novel internalizing tumor cellsurface receptors which will be useful therapeutic targets. If thisapproach proves useful, we plan on applying it to primary tumor cellsand DCIS. We also intend to evaluate 3TF5 (ErbB2 binding scFv which isinternalized faster than C6.5) for liposome targeting. It is possiblethat it will be more effective than C6.5

In addition, the experiments demonstrate that methodologies forincreasing antibody affinity in vitro to values not previously achievedin vivo. We have applied these methodologies to generate novel ErbB2binding scFv.

Example 5 Epitope Mapping of F5 and C1

Two unique phage antibodies were identified which were internalized bySKRB3 cells (F5 and C1 described above). Neither of these phage wereisolated when the same library was selected on recombinant ErbB2. Todetermine why, the K_(d) of F5 (3.2×10⁻⁷ M) and C1 (K_(d)=1.0×10⁻⁶ M)were measured. These K_(d) are significantly higher than the K_(d)measured for four of the scFv selected on recombinant ErbB2 (K_(d)=0.1to 0.65 nM). The higher K_(d) internalizing phage antibodies would haveto compete with the lower K_(d) non-internalizing phage antibodies forselection on recombinant ErbB2 and were likely lost during the selectionprocess. Since antibodies which are internalized as monomers are likelyto be rare, and since there will be many more phage antibodies of loweraffinity than higher affinity in a library, it is not surprising thatthe internalizing antibodies are of high K_(d). Since antibodies whichare internalized are likely to be rare, we hypothesized that it waslikely that F5 and C1 recognized the same epitope. This was confirmedusing a competition assay (FIG. 2). Thus as hypothesized, F5 and C1recognize the same epitope, and a different epitope than C6.5. Using thesame assay, we confirmed that F5 and C1 recognize a different epitopethan the Genentech anti-ErbB2 antibody 4D5 (when humanized known asHerceptin

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-22. (canceled)
 23. A method of specifically delivering an effectormolecule to a cell bearing a c-erbB2 receptor, said method comprising:providing a chimeric molecule comprising said effector molecule attachedto an internalizing antibody that specifically binds to the c-erbB2receptor, wherein said antibody specifically binds to a c-erbB2 epitopeas bound by F5 (SEQ ID NO: 1) or C1 (SEQ ID NO: 2); and contacting saidcell bearing a c-erbB2 receptor with said chimeric molecule, wherebysaid chimeric molecule specifically binds to said cell.
 24. The methodof claim 23, wherein said effector molecule is selected from the groupconsisting of a cytotoxin, a label, a radionuclide, a drug, a liposome,a ligand, and an antibody.
 25. The method of claim 23, wherein saidchimeric molecule is a fusion protein.
 26. The method of claim 23,wherein said cell is a cancer cell.
 27. The method of claim 26, whereinsaid cancer cell is a breast cancer cell.
 28. The method of claim 23,wherein said antibody shares at least 70% sequence identity with theamino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and wherein saidantibody has a binding affinity for c-erbB2 receptor of at least 10 M.29. The method of claim 23, wherein the amino acid sequence of saidantibody differs from the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 2 by no more than 30 residues.
 30. The method of claim 23, whereinsaid antibody comprises a complementarity determining region (CDR) ofSEQ ID NO:
 1. 31. The method of claim 23, wherein said antibodycomprises a complementarity determining region (CDR) of SEQ ID NO: 2.32. The method of claim 23, wherein said antibody has the amino acidsequence of SEQ ID NO:
 1. 33. The method of claim 23, wherein saidantibody has the amino acid sequence of SEQ ID NO:
 2. 34-44. (canceled)45. A nucleic acid encoding an antibody that specifically binds to theepitope of c-erbB2 receptor bound by F5 (SEQ ID NO: 1) or C1 (SEQ ID NO:2).
 46. The nucleic acid of claim 45, wherein said nucleic acid encodesan amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 1 having conservative substitutions, and SEQID NO: 2 having conservative substitutions.
 47. The nucleic acid ofclaim 45, wherein nucleic acid encodes an antibody that shares at least70% sequence identity with the amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 2 and wherein said antibody has a binding affinity forc-erbB2 receptor on cells of at least 10 μM.
 48. The nucleic acid ofclaim 45, said nucleic acid encodes an amino acid sequence of saidantibody that differs from the amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 2 by no more than 30 residues.
 49. The nucleic acid of claim45, wherein said nucleic acid encodes a complementarity determiningregion (CDR) of SEQ ID NO:
 1. 50. The nucleic acid of claim 45, whereinsaid nucleic acid encodes a complementarity determining region (CDR) ofSEQ ID NO:
 2. 51. The nucleic acid of claim 45, wherein said nucleicacid encodes the amino acid sequence of SEQ ID NO:
 1. 52. The nucleicacid of claim 45, wherein said nucleic acid encodes the amino acidsequence of SEQ ID NO:
 2. 53-54. (canceled)